Vol.2 No.1
2022 December
ISSN 2436-4975

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OBTURATION

メタルフリー材料・接着・IOS の 融合 で 実現 する
次世代臨床コンセプト「オクルーザルベニア」の理論的背景 と 臨床ステップ を 提示

オクルーザルベニアとは？
歯肉縁上へのフィニッシュライン設定による最小限の侵襲の後に，歯冠修復材料に
レジン系材料やジルコニア，二ケイ酸リチウムなどのセラミックスを用いて接着により支台歯に固定．次世代の技術を活用した，新しい歯冠修復法のコンセプトです．

歯界展望 別冊D e n t a l

O u t l o o k

進化したメタルフリー材料・接着・I OSの融合で実現する
次世代臨床コンセプト

本書には，オクルーザルベニアの何が書いてある？
オクルーザルベニアのエビデンスから実際の臨床でのポイントまで網羅しました．
そのなかでも，
オクルーザルべニアを安全に使用するための最も重要な要素として，「支台歯形成」と「接着術式」については特に詳しく説明しています．

新谷明一・三浦賞子・小泉寛恭・二瓶智太郎・峯宮崎真至・海渡智義 著

篤史

オクルーザルベニアの実際（ 7 6 の歯冠修復）
左：修復形態（被覆範囲）の決定後，支台歯形成．フィニッシュラインを歯肉縁上に設定．
右：シリカ系セラミックスを用いて CAD/CAM により歯冠修復物を製作．接着により支台歯に固定．

https://www.ishiyaku.co.jp/（写真提供：海渡智義先生， 本書 Chapter 10 より）〒 113-8612 東京都文京区本駒込 1-7-10
TEL03-5395-7630 FAX03-5395-7633 https://www.ishiyaku.co.jp/■ A4 判変／ 144 頁／カラー■ 定価 6,930 円（本体 6,300 円＋税 10％）■ 注文コード：351220

編著

OPERATIVE DENTISTRY, ENDODONTOLOGY AND PERIODONTOLOGY
Vol. 2, No. 1

CONTENTSDECEMBER 2022

Original Articles
Effect of the Thickness Ratio of Core Build-up Composite Resin to Root Dentin
on the Strength of Abutment Construction
Yoh KOBAYASHI, Miki SEKIYA, Keisuke SAIGUSA,
Munehiro MAEDA and Masaru IGARASHI （ 1 ）Effects of Sodium Copper Chlorophyllin on Human Gingival Fibroblasts in vitro
Kosuke MARUYAMA, Kayo SATO, Susumu NAKATSU,
Takuya ASADA, Hidetake FUJINAKA, Yoshitaka YANO
and Soh SATO （ 9 ）Three-year Retrospective Follow-up of Cases of Root Canal Obturation
with a Bioactive Glass-based Root Canal Sealer
Ayako WASHIO, Hiroki MIURA, Miki SUEMATSU,
Kazumasa MURATA, Ryosuke AIHARA, Masataka FUJIMOTO,
Ai ORIMOTO and Chiaki KITAMURA （ 16 ）Influence of Light Irradiation Modes on the Polymerizationof Light-curing Resins in a Root Canal Model
Kazumasa MURATA, Hiroki MIURA, Hiroshi IKEDA,
Shinji YOSHII, Ayako WASHIO and Chiaki KITAMURA （ 25 ）In vivo Assessment of Tissue Compatibility of Root Canal Sealer
Containing Surface Pre-reacted Glass-ionomer Fillers
Saori TANAKA, Hirofumi MIYAJI, Kayoko MAYUMI,
Yukimi KANEMOTO and Erika NISHIDA （ 33 ）Maillard Reaction Product of Rare Sugar Allulose Decreases
Bacteria-derived and Chemically-prepared Hydrogen Sulfide
Shigeru YAMAMOTO, Nao TANIGUCHI, Jun TAKATORI,
Junko HATAKEYAMA, Sami OMAGARI, Kazuhiko YAMADA,
Takao HIROFUJI and Masahiro YONEDA （ 40 ）Study on Sealability of Dentinal Tubules Irradiated by Nd: YAG Laserwith TiO2 Using a Model of Hypersensitive Dentin
Tomohiro NAKATA, Kenzo YASUO, Yuto MORIKAWA,
Naohiro IWATA, Hiroaki TANIMOTO, Kazushi YOSHIKAWA
and Kazuyo YAMAMOTO （ 49 ）Application of Intraoral Scanners in Dental Health Guidance―Quantification and Visualization of Marginal Gingival Changes―
Akina TANI, Akane OMORI, Shizuo HIGUCHI
and Kazutoshi KAKIMOTO （ 60 ）Submission Guidelines and Guidance
Publishedby
THE JAPANESE SOCIETY OF CONSERVATIVE DENTISTRY（JSCD）
c/o Oral Health Association of Japan（Kōkūhoken kyōkai）
1‒43‒9, Komagome, Toshima-ku, Tokyo 170‒0003
Japan（ 72 ）

Editorial Board of Operative Dentistr y, Endodontology and Periodontology
Editor-in-Chief
Hideki SHIBA

Graduate School of Biomedical and Health Sciences, Hiroshima University

Associate Editor
Hidefumi MAEDA

Kyushu University Faculty of Dental Science

Editorial Board

Atsushi KAMEYAMA
Matsumoto Dental University

Kazuo KITAMURAThe Nippon Dental University

Satoshi HIRAYAMA
Nihon University School of Dentistry at Matsudo

Takashi MURAMATSU
Tokyo Dental College

Takashi YAEGASHI
School of Dentistry, Iwate Medical University

Kazuyo YAMAMOTO
Osaka Dental University

Matsuo YAMAMOTO
Showa University School of Dentistry

Hiromichi YUMOTO
Tokushima University Graduate School of Biomedical Sciences

Kunihiko YOSHIBA
Niigata University Graduate School of Medical and Dental Sciences

Atsutoshi YOSHIMURA
Nagasaki University Graduate School of Biomedical Sciences

Masahiro YONEDA
Fukuoka Dental College

Editorial Secretar y
Katsuhiro TAKEDA

Graduate School of Biomedical and Health Sciences, Hiroshima UniversityDec, 2022

1

Original Article

ODEP 2（1）：1～8，2022

Effect of the Thickness Ratio of Core Build-up Composite Resinto Root Dentin on the Strength of Abutment Construction
Yoh KOBAYASHI, Miki SEKIYA, Keisuke SAIGUSA,
Munehiro MAEDA and Masaru IGARASHI
Department of Endodontics, The Nippon Dental University School of Life Dentistry at Tokyo

Abstract
Purpose: After root canal treatment, the tooth is restored with an abutment, for which core build-up resinsproviding adhesiveness have come into use. This study examined the effect of the thickness ratio of the composite resin used for construction and the thickness of the root dentin on the strength of the construction.
Methods: This research used four core build-up resins: 1）Clearfil DC Core Automix ONE（DC）, 2）BeautiCore Post Paste（BC）, 3）Filtek Fill and Core Flowable Restorative Plus（FT）, and 4）Estecore（EC）. First,164 experimental specimens of 2.0 mm×2.0 mm×14.0 mm, with dentin thicknesses of 0.5（D0.5）, 1.0（D1.0）,
1.5（D1.5）, and 2.0 mm（D2.0）, were prepared from bovine teeth, which were subjected to a three-point bending test in a universal testing machine, and the fracture load was recorded. Second, 40 extracted human single-root teeth were used to perform compressive strength tests of dentin-resin composites. The root side of
the tooth was cut to a length of 15 mm from the root apex, and root dentin with the post space of 1/3 width（1/3 P）, which is a standard post formation, was used as the control group. Root dentin widths of 2/3（2/3P）, 3/4（3/4 P）, and 4/5（4/5 P）, and human extracted teeth without core build-up（S）were used as theexperimental groups. A universal testing machine was used to apply a static load in the tooth axial direction
at a crosshead speed of 1.0 mm/min, and the fracture load was recorded.
Results: The bending strength of FT was significantly lower than those of BC and EC in the D0.5 group（p＜0.05）. The bending strength of FT was significantly lower than that of EC in the D1.0 and D1.5 groups（p＜0.05）.
The bending strengths of the four core build-up resin groups were significantly greater than that of the S
group, which only underwent root canal shaping.
The highest force required for fracture among the four groups was 41.1±10.6 MPa in the 3/4 P group.
This was followed by 39.1±8.8 MPa in the 1/3 P group, 37.5±9.5 MPa in the 4/5 P group, 33.4±4.1 MPa in
the 2/3 P group, and 15.3±7.3 MPa in the S group.
Conclusion: It is suggested that construction using a core build-up resin offers similar compressive
strength as conventional abutment construction based on 1/3 width, even if the remaining tooth root is thinner.
Key words: core build-up resins, bending strength, compressive strength

Corresponding author: Yoh KOBAYASHI, Department of Endodontics, The Nippon Dental University School of Life Dentistryat Tokyo, 1‒9‒20, Fujimi, Chiyoda-ku, Tokyo 102‒8159, Japan
TEL: ＋81‒3‒3261‒5698, FAX: ＋81‒3‒5216‒3718, E-mail: y-kobayashi2118002@tky.ndu.ac.jp
Received for Publication: January 7, 2022/Accepted for Publication: January 18, 2022
J-STAGE advance published date: October 14, 2022
DOI: 10.11471/odep.2022-001

ODEP Vol. 2, No. 1

2

addressed the effects on the strength of abutment con-Introduction

struction when the ratio of the thickness of the composite resin to remnant root dentin is changed.

After root canal treatment, if the obturated tooth is

Therefore, this study examined the effects of theleft untreated for a long period of time, the pulp cham-thickness of the composite resin used for construction

ber becomes contaminated with bacteria and the inter-and the thickness of the root dentin on the strength of

facial gap between the filling material and the root

the construction.

canal wall causes bacterial coronal leakage1‒4）, resulting
in the development of periapical periodontitis. Endodon-

Materials and Methods

tically treated teeth are constructed with the abutment
construction to fill the defect and restore the functional

1 ．Experiment 1: Bending strength test based onform because the dentin of the root canal wall is

dif ferences between dentin and resin thick-removed and thinned during root canal preparation. At

nessthat time, it is important to maintain tooth strength,

1 ）Experimental core build-up resin materials

and metal materials have generally been used. In gen-This study used four core build-up resins for abut-eral, the core build-up requires a ferrule of over 1 mm

ment construction:（1）Clearfil DC Core Automix ONE

and remaining tooth substance of ≥1 mm from the cer-（DC）
（Kuraray Noritake Dental, Tokyo, Japan）,（2）vical region to the crown side as conditions5,6）. Further,

BeautiCore Post Paste（BC）（Shofu, Kyoto, Japan）,（3）the thickness of the post is based on the standard of not

Filtek Fill and Core Flowable Restorative Plus（FT）

exceeding 1/3 of the maximum width of the tooth（3M, Saint Paul, MN, USA）
, and（4）Estecore（EC）

root7）, and the residual amount of healthy tooth sub-（Tokuyama, Tokyo, Japan）. The tooth surface treat-stance is important. However, metallic materials havement agent specified by each manufacturer was used

the problem that their elastic modulus differs signifi-

for adhesion treatment of the tooth substance（Table

cantly from that of dentin, causing stress on the luting1）.

cement . In addition, a removable wax pattern is2 ）Preparation of an adhesive model

required during the process of manufacturing the metal

One hundred extracted bovine single-root teeth were8）

core, so a large amount of dentin needs to be removed

sliced into 2.0 mm thicknesses using a low-speed preci-

to avoid leaving an undercut.

sion cutting machine（ISOMET 1000; Buehler, LakeIn recent years, core build-up resins that have adhe-Bluff, IL, USA）
, and 200 test pieces of 2.0 mm width×

siveness to dentin and have similar physical character-

14.0 mm length were cut out. From these 200 samples,

istics to those of dentin have been developed. These are

164 samples were selected for the experiment.

commercially available in a chemical-cured type, and

The thickness of the test pieces was adjusted by pol-dual-cured（light- and chemical-cured）type. The coreishing the surfaces with open dentin tubules with #600

built up using such adhesive materials provides excel-

water-resistant abrasive paper until the thickness waslent marginal sealing ability and can prevent coronal

0.5 mm, 1.0 mm, and 1.5 mm, respectively.

leakage. In particular, the direct resin core build-up can

3 ）Preparation of resin-bonded sections

be applied with or without undercut in the tooth cavity.

A slot of 2.0 mm width×2.0 mm depth×25.0 mm

In cases where the metal post crown falls off, second-

length cavity was made by two L-shaped metal plates

ary caries often progresses in the dentin around the（Fig. 1）, and a dentin piece that had been treated for

dowel post. When the softened dentin of the root canalbonding according to the procedure specified by eachwall is completely removed, many cases only show one

manufacturer was inserted into the bottom of the slot.

layer of thin dentin remaining near the root surface. In

The four core build-up resins shown in Table 1 wereclinical practice, most cases of endodontic re-treatment

embedded in the adhesive surface of the dentin piece,

require removal of prosthetic devices, posts, and caries,

and while manually pressing on the glass, irradiation

resulting in less residual dentin. Under such circum-

was performed for 10 s with a light irradiator（G-Light

stances, many cases do not match the basic morphology

Prima Ⅱ Plus; GC, Tokyo, Japan）. The cured experi-of conventional cavities. So far, no reports havemental piece was taken out from the metal frame, andDec, 2022

Effect of Thickness of Core Resin on Abutment Construction
Table 1

3

Compositions of each core build-up resin used in this study

Materials（Maker）

Composition

Adhision

Clearfil DC Core Automix ONE（Kuraray Noritake Dental）

Paste A：Bis-GMA, hydrophobic aliphatic dimethacrylate,hydrophilic aliphatic dimethacrylate, hydrophobic aromatic
Clearfil Universal Bond
dimethacrylate, filler, dl-CQ, initiators, pigments
Quick ER
Paste B：TEGDMA, hydrophilic aliphatic dimethacrylate,hydrophobic aromatic dimethacrylate, filler, accelerators

BeautiCore Post Paste（Shofu）

Glass powder, Bis-GMA, TEGDMA, UDMA

BeautiBond Xtreme

Filtek Fill and Core Flowable
Restorative Plus（3M）

Glass powder, Bis-GMA, TEGDMA, UDMA

Scotchbond Universal
Adhesive

Estecore（Tokuyama）

Paste A：Silica zirconia filler, Bis-GMA, TEGDMA, Bis-MPEPP
Paste B：Silica zirconia filler, Bis-GMA, TEGDMA, Bis-MPEPP, Bondmer Lightless
peroxides, camphorquinone, radical amplifiersABa

2.0 mmb
25 .0mm
The template used to prepare test pieces
A：Structure of the template. a is L-shaped plate, b is base plate. B：The combined state.
Fig. 1the excess resin was polished and removed with

2 ．Experiment 2: Compressive strength test ofwater-resistant silicon carbide paper #600 to prepare an

dentin resin complex using extracted humanexperimental piece measuring 2.0 mm×2.0 mm×14.0

teeth

mm. A specimen made of resin alone was used as theAfter obtaining approval from the Nippon Dental

control（2.0）. Three types of samples were used as

University Ethics Committee（approval number NDU-

experimental groups: D0.5 group（0.5 mm dentin thick-T2019-44）, 40 extracted human single-root teeth col-ness）
, D1.0 group（1.0 mm dentin thickness）, and D1.5

lected from healthy subjects aged 15 to 40 years oldgroup（1.5 mm dentin thickness）. Fourteen specimens

were used in the experiment.

of each group were used in the experiment. R2.0 was

Mandibular premolars were used as samples because

consisted of whole resin. The specimens with a dentin

they are easily extracted for orthodontic convenience

thickness of 2.0 mm were used as the D2.0 group forand have less caries. After access opening of the pulp

reference（Fig. 2）.

chamber, the root canals were shaped using the stan-4 ）Three-point bending test

dardized preparation technique with a size #50 K file

The produced sample piece was subjected to a three-（MANI Co., Tochigi, Japan）. Size #50 master points

point bending test at a crosshead speed of 1.0 mm/min（GC, Tokyo, Japan）and accessory points（GC）, and a

on a universal testing machine（Autograph AGS-X;

root canal sealer（Canals; Showa Yakuhin Kako Co.,

Shimadzu, Tokyo, Japan）, and the fracture load wasTokyo, Japan）were used for the lateral condensation

recorded（Fig. 3）.

method. The crown side was then cut so that the root
length was 15 mm from the apex. A diamond bar（Matsukaze Diamond Point FG-C105RX; Matsukaze, Tokyo,

ODEP Vol. 2, No. 1

4
14.0 mm

2.0 mm
1.5 mmRD
D0.5

0.5 mm

2.0 mmD2.0

1.0 mm
1.0 mmD1.0

2.0 mmR2.0

0.5 mm
1.5 mm
D1.5

Fig. 2 Schematic diagram of each plate specimen
R：Resin, D：Dentin, D0.5：0.5 mm dentin thickness, D1.0：1.0 mm dentin thickness, D1.5：
1.5 mm dentin thickness, D2.0：2.0 mm dentin thickness, R2.0：2.0 mm resin thickness

bending test, the bending strength of EC was increasedLoaddepending on its thickness. This property was excluded
6.0 mmA

because it was considered to affect the results of thisexperiment. FT was weaker in intensity than the other6.0 mm
2.0 mmSpecimengroups. In this study, there was no significant difference in the three-point bending strength between DC
and BC. However, DC has been reported in previousBB

studies to have superior bond strength9）. Therefore, DC
was employed in the experiments for compressive
strength.
Then, DC was filled into the tooth cavity and cured

Schematic diagram of thethree-point bending test（head speed：1.0 mm/min）with a light irradiator. Each sample was stored in an

A：indenter, B：load bearing platform

tester was used to apply static load along the tooth axis

Fig. 3incubator at 37℃ for 24 h. After storage, a universal
direction at a crosshead speed of 1.0 mm/min, and themeasured fracture load was recorded（Fig. 5）.

Japan）was equipped in the micromotor of a cus-

3 ．Statistical analyses

tom-made preparing device（Nihon Mecc, Tokyo, Japan）

Statistical analysis software（SPSS version 25; IBM,and used for post preparation of 1/3 width of the root

North Castle, NY, USA）
was used for statistical analysis.

（1/3 P）, from the apex to 5.0 mm according to the stan-

Tukey’s test was used for the three-point bending test

dard post formation（control group）. The experimental

in bovine teeth, and one-way analysis of variance andgroups（n＝10）were the four groups of the 2/3 forma-

Tukey’s multiple comparison test were used for thetion group（2/3 P）, the 3/4 formation group（3/4 P）, andcompressive strength test in extracted human teeth.

the 4/5 formation group（4/5 P）, and the group in

The differences in the failure loads between the condi-

which only canal shaping was performed（S）（Fig. 4）.

tions were tested for significance at the 5％ level.

The inside of the tooth cavity was treated with an etching agent（K Etchants GEL, Kuraray Noritake Dental,

Results

Tokyo, Japan）for 40 s, washed with water and dried,then the tooth surface was treated with Clearfil Univer-

1 ．Three-point bending test

sal Bond Quick ER（Kuraray Noritake Dental）as

The results of three-point bending tests of the five

instructed by the manufacturer. In the three-point

groups in which the thicknesses of bovine dentin wereDec, 2022

Effect of Thickness of Core Resin on Abutment Construction
A

B

CONTROL
1/3

a

2/3

1/3

Post cavity

a

3/4P

b

4/5P

c

4/5

Root dentin

15.0 mm

2/3P
3/4

1/3
b

5

GP＋Sealer
5.0 mmSchematic diagram of the dentin and resin complex using
extracted human teeth
A ：Control. a：vertical view, b：horizontal view：the sample removed to
1/3 of root width
B ：Horizontal views of various samples. a：the sample removed to 2/3 ofroot width, b：the sample removed to 3/4 of root width, c：the sample removed to 4/5 of root width

IndenterSpecimen

Fig. 5 Schematic diagram of static load test（head speed：
1.0 mm/min）
The specimen is placed in a cylindrical jig with the root
apex upward, and the indenter is used to apply pressure ata head speed of 1.0 mm/min until the specimen is destroyed.

Three-point bending strength（MPa）Fig. 4

250
＊

200

＊
＊
＊

＊

D0.5
D1.0
D1.5

150

R2.0

100
50
0

Fig. 6

DC

BC

FT

EC

Measured values of three-point bending
strength for each group（＊：p＜0.05, n＝14）

changed using the four core build-up resins are shownin EC at 168.8±38.8 MPa, followed by BC at 142.5±30.1

in Fig. 6. Bending strength for the D0.5 group was high-MPa, DC at 140.3±22.9 MPa, and FT at 118.6±29.0

est in BC at 134.7±30.1 MPa, followed by 133.0±41.5

MPa. Bending strength in FT was again significantly

MPa in EC, 122.5±30.9 MPa in DC, and 100.0±23.9

lower than that in EC（p＜0.05）. Bending strength for

MPa in FT. Bending strength of FT was significantly

the R2.0 group was highest in EC at 164.16±34.2 MPa,l o w e r t h a n t h a t o f B C o r E C（p＜0 . 0 5）
. Bendingfollowed by 130.73±13.7 MPa in DC, 124.68±10.6 MPa

strength for the D1.0 group was highest in EC at 144.8

in FT, and 121.54±17.8 MPa in BC. Bending strength±34.0 MPa, followed by 128.8±29.7 MPa in DC, 127.4±

for the D2.0 group was 220.9±56.3 MPa.

36.2 MPa in BC, and 103.2±20.5 MPa in FT. Bending

2 ．Compressive strength test

strength was significantly lower in FT than in EC（p＜The results of the load measured by applying a static

0.05）. Bending strength for the D1.5 group was highest

load using four groups of core build-up teeth by chang-

ODEP Vol. 2, No. 1

Breaking load value（MPa）

6
＊

60

＊

50

and resin, but in recent years fewer cases of human＊

tooth extraction have been performed due to advances
in dental care, making collection of samples more diffi-＊

cult. Bovine teeth are relatively easily available, and40

previous studies have shown the effectiveness of using
bovine teeth as a substitute for human teeth10‒13）.

30

A minimum of 12 mm is required for a plate-shaped

20

sample for a three-point bending test, but cutting out 12100

mm from a human dentin tooth root in an anatomical
4/5 P

3/4 P

2/3 P

1/3 P

S

Measured values of fracture load foreach group（＊：p＜0.05, n＝10）
1/3P：post preparation of 1/3 width of theroot（control group）, 2/3P：2/3 formation group,
3/4P：3/4 formation group, 4/5P：4/5 formation
group
S：group in which only canal shaping was performed
Fig. 7

form is difficult. It was easier to prepare test pieces forthe three-point bending test using bovine teeth, as they
are readily prepared, can be adjusted to a length of 12mm or more, and show similar adhesive strength tohuman teeth. In a comparison among the four materials, the bending strength of DC, BC, and FT increased
as the thickness increased to D1.0 and D1.5 comparedto D0.5, but no significant difference was observed. On
the other hand, in EC, strength differed with changes indentin thickness between the D0.5 and the D1.5 groups,
indicating that strength depended on the dentin. In theing the thickness of the core build-up resins are shownpresent study, the strength of other experimental

in Fig. 7.

groups also showed a tendency to increase with

The highest force required for fracture among theincreasing dentin thickness.

four groups was 41.1±10.6 MPa in the 3/4 P group.

In addition, it was reported that EC has stronger

This was followed by 39.1±8.8 MPa in the 1/3 P group,

adhesive strength than other core resins8）. In EC, it is37.5±9.5 MPa in the 4/5 P group, 33.4±4.1 MPa in theconsidered that the degree of increase in strength rose

2/3 P group, and 15.3±7.3 MPa in the S group.

further with the increase in dentin thickness. The

However, there was a significant difference between

three-point bending strength of DC, BC, and FT tended

the group of teeth in which only formation was done

to increase as the thickness of the dentin increased

and the group in which each formation was done.

from 0.5 to 1.0 and 1.5, but there was no significant difference. The strength of FT was lower than that of theDiscussion

other groups. It is speculated that FT has a wide range
of applications such as filling of cavities in addition to

For endodontically treated tooth, core build-up isabutment construction. The D2.0 group showed higher

required to manufacture the prosthetic device. The

values than the other groups. It is speculated that theeffect of the thickness of the core build-up resins on

structure of dentinal tubules in dentin is different fromtooth strength needs to be determined. Therefore, athe three-dimensional structure of resin, which contrib-

comparative study was conducted on the effect of theuted to the dispersion of force.

ratio of the thickness of the core build-up resins forWith the three-point bending test, the average

construction and the thickness of the residual root den-strength of the dentin plate-shaped test pieces with dif-tin on the strength of the construction body.

ferent thicknesses was 100‒168 MPa, similar to the

In the experiment, extracted bovine teeth were usedvalue of 106‒173 MPa shown for the core build-up res-to allow easy creation of standard plate-shaped samples,ins in a previous study14）. There was a difference inand the physical properties of the adhesive system thatbending strength between the D0.5 and D1.5 groups ofis currently widely used in clinical practice and core

EC. Since the R2.0 group of EC had the highest

build-up resins were compared. In this study, an adhe-strength among the four groups, it is considered thatsive sample was prepared from pieces of bovine dentin

even if the thickness of EC became thin, it contributed

Dec, 2022

Effect of Thickness of Core Resin on Abutment Construction

7

to the bending strength of the resin.
Our results showed that the adhesion between theConclusions

resin and dentin plate had a similar strength as thecore build-up resins alone, regardless of the thickness.

The following conclusions were reached:This indicates that the strength is guaranteed if an

1．The results of the three-point bending test

appropriate resin thickness can be achieved, even in

showed that there was no difference in the bending

tooth roots with a thinned tooth structure. Therefore,

strength of each resin except EC when the thickness ofeven a tooth in which the healthy tooth structure isdentin was changed.

thinned by caries around the metal post has sufficient

2．There was no difference in the compressive

strength and resin can be applied. Also, in previous

strength of extracted human roots loaded with resin at

research, samples of the core build-up resins alone weredifferent thicknesses of dentin.

used for the three-point bending test, but the present
study succeeded in producing a composite sample of

3．EC had significantly greater three-point bending
strength than FT in all dentin thicknesses.

bovine teeth and resin, and the numerical value of the

These findings suggest that construction using core

bending strength was also similar to those of previous

build-up resins offers the same strength as conventional

studies15,16）. Since those results were close to the pres-abutment construction based on 1/3 of the tooth struc-ent results, reproducing the three-point bending test inture removed, even if the tooth root is thin.

more clinical situations seems feasible.
In the compressive strength testing of the dentin

Conflict of interest statement

resin complex using extracted human teeth, no differ-

There are no conflicts of interest to disclose regarding
this paper.

ence in strength was found regardless of the amount ofdentin removed. The results showed that the mechanical properties of the composite resin used for abutment
construction in recent years are very close to those ofdentin, and the adhesion between dentin and resin
17,18）

results in an integrated composite. In addition, thestrength against vertical fracture is greatly increased
when the interior is replaced with resin rather thanleaving the tooth without core build-up resins. In addition, the formation comprising 1/3 of the root width, as
the principle of post formation, does not necessarily
represent a limitation to the use of core build-up resins,and it is not a problem even if the thickness of root
dentin is thinner due to caries or excessive root canalenlargement. We clarified that the abutment can beconstructed without diminishing the strength of thethinned tooth root. Even in the apexification of a toothwith incomplete root formation, where treatment iscompleted with a thin residual tooth substance, tooth
fracture has become a problem during long-term follow-up. In such cases, even if the tooth substance isthin, it may be possible to maintain strength by appropriately treating the tooth surface and filling the cavity
with core build-up resins.

References
1）Fukushima S, Tsubota Y. Postoperative clinical evaluation of posts and cores. J Jpn Prosthodont Soc 2001; 45:660‒668.
（in Japanese）
2）Mine A, A systematic review of preventive measuresagainst root fractures“in 2013”. Ann Jpn Prosthodont
2014;
（6）
: 26‒35.
（in Japanese）
3）Tsubota Y, The present of restoration of endodonticallytreated teeth. Ann Jpn Prosthodont 2017;（9）: 94‒100.
（inJapanese）
4）Tabassum S, Khan FR. Failure of endodontic treatment:
The usual suspects. Eur J Dent 2016; 10: 144‒147.
5）Juloski J, Radovic I, Goracci C, Vulicevic ZR, Ferrari M.
Ferrule effect: a literature review. J Endod 2012; 38:
11‒19.
6）Peroz I, Blankenstein F, Lange KP, Naumann M. Restoring endodontically treated teeth with posts and cores―a review. Quintessence Int 2005; 36: 737‒746.
7）Lloyd PM, Palik JF. The philosophies of dowel diameter
preparation: a literature review. J Prosthet Dent 1993;
69: 32‒36.
8）Jung SH, Min KS, Chang HS, Park SD, Kwon SN, Bae
JM. Microleakage and fracture patterns of teeth
restored with different posts under dynamic loading. J
Prosthet Dent 2007; 98: 270‒276.
9）Ariyoshi M, Nikaido T, Okada A, Foxton RM, Tagami J.

ODEP Vol. 2, No. 1

8
Dentin bond strengths of three adhesive/composite

2021; 14: 2099.

core systems using different curing units. Dent Mater J;
2008, 27: 187‒194.

14）Wada Y, Miyake-Aoki K, Kameyama Y, Ohashi K,
Midono T, Oshikawa A, Shimoyama K, Nihei T. Flex-

10）Krifka S, Börzsönyi A, Koch A, Hiller KA, Schmalz G,
Friedl KH. Bond strength of adhesive systems to dentin
and enamel―human vs. bovine primary teeth in vitro.
Dent Mater 2008; 24: 888‒894.
11）Camargo CHR, Siviero M, Camargo SEA, Oliveira SHG,
Carvalho CAT, Valera MC. Topographical, diametral,and quantitative analysis of dentin tubules in the root
canals of human and bovine teeth. J Endod 2007; 33:422‒426.
12）Soares FZM, Follak A, Rosa LS, Montagner AF, Lenzi
TL, Rocha RO. Bovine tooth is a substitute for human
tooth on bond strength studies: A systematic review
and meta-analysis of in vitro studies. Dent Mater 2016;
32: 1385‒1393.
13）Shimizu K, Satoh T, Shinkai K, Evaluation of restorativetechniques for vertically fractured roots. Materials

ural strength and adhesion of resin composite for constructing tooth foundation. Bull Kanagawa Dent Coll
2019; 54‒1: 1‒9.
（in Japanese）
15）Spinhayer L, Bui ATB, Leprincea JG, Hardy CMF. Core
build-up resin composites: an in-vitro comparative study.
Biomater Investig Dent 2020; 7: 159‒166.
16）Irie M, Maruo Y, Nishigawa G, Yoshihara K, Matsumoto
T. Flexural strength of resin core build-up materials:
Correlation to root dentin shear bond strength and pullout force. Polymers 2020; 12: 2947.
17）Belli S, Eraslan O, Eskitascioglu G, Karbhari V. Monoblocks in root canals: a finite elemental stress analysis
study. Int Endod J 2011; 44: 817‒826.
18）Tay FR, Pashley DH. Monoblocks in root canals: a hypothetical or a tangible goal. J Endod 2007; 33: 391‒398.

Dec, 2022

9

Original Article

ODEP 2（1）：9～15，2022

Effects of Sodium Copper Chlorophyllinon Human Gingival Fibroblasts in vitro
Kosuke MARUYAMA1, Kayo SATO2, Susumu NAKATSU2, Takuya ASADA2,
Hidetake FUJINAKA2, Yoshitaka YANO2 and Soh SATO1,3
1

Department of Periodontology, The Nippon Dental University School of Life Dentistry at Niigata
2

Personal Health Care Products Research Laboratories, Kao Corporation

3

Niigata Hospital, Comprehensive Dental Care, The Nippon Dental University

Abstract
Purpose: Sodium copper chlorophyllin（SCCP）is known to have various effects such as antiallergic andtissue-activating effects, and in recent years is expected to be effectively used in the medical field. Moreover,in the field of dentistry, it is used for treating periodontal disease and trauma. Although there are many
reports on the effects of SCCP, few reports have examined how it works. In this study, to elucidate theeffects of SCCP on periodontal tissue, we studied physiological changes in vitro in human gingival fibroblasts（HGF）exposed to SCCP.
Methods: Human gingival fibroblasts were cultured in 15％ FBS-containing DMEM. The cells were
exposed to SCCP（5 nM, 10 nM, 100 nM, 1μM, 10μM）, and cell proliferation and mitogens were examined.
Results: Cell proliferation increased significantly with 100 nM SCCP, and an increase in mitotic factors that
promoted cell proliferation was observed.
Conclusion: The results suggested that 100 nM SCCP stimulates cell division factors of human gingival
fibroblasts and promotes cell proliferation, thereby promoting wound healing.
Key words: human gingival fibroblast, sodium copper chlorophyllin, wound healing, periodontal tissue

Corresponding author: Soh SATO, Department of Periodontology, The Nippon Dental University School of Life Dentistry at
Niigata, 1‒8 Hamaura-cho, Chuo-ku, Niigata 951‒8580, Japan
TEL: ＋81‒25‒267‒1500（ext. 3338）, FAX: ＋81‒25‒233‒3983, E-mail: s-sato@ngt.ndu.ac.jp
Received for Publication: January 14, 2022/Accepted for Publication: May 18, 2022
DOI: 10.11471/odep.2022‒002

ODEP Vol. 2, No. 1

10odontal tissue, we studied physiological changes in vitro

Introduction

in human gingival fibroblasts（HGF）exposed to SCCP.

Sodium copper chlorophyllin（SCCP）is an alkaline

Materials and Methodshydrolysis product of copper chlorophyll, which contains Cu in place of Mg in the center of the chlorophyll

1 ．Sodium copper chlorophyllin

structure（Fig. 1（A））
. It is an assembly of components

The SCCP（Lot 60221）used in the experiment waswith similar chlorophyll skeletons, rather than a single

supplied by TAMA BIOCHEMICAL Co.（Tokyo,

component1）. SCCP has various effects including antial-

Japan）. In order to identify the primary small molecule

lergic, tissue-activating, and deodorizing effects2,3）. With

constituents contained in the supplied SCCP, mass anal-

expectation of these effects, it has been applied in theysis was performed using a liquid chromatograph time-

field of medicine. For peptic ulcer, orally administeredof-flight mass spectrometer equipped with a photodiode

SCCP exerts excellent healing promoting effect byarray. The measurement conditions were in accordance

reducing pepsin activity in the gastrointestinal tract

with the method of Simaremare et al15）.

and inducing the proliferation of fibroblasts in connec-

2 ．Human periodontal tissue

4）

tive tissue . For dermatitis and urticaria, topically

HGF were obtained using the outgrowth method

applied SCCP exerts antiallergic effect and promotes

from the gingival tissue on a patient’s teeth removed

healing5）. In the field of dentistry, SCCP is used for end-

for therapeutic reasons at the Hospital（a female withodontic treatment due to its antibacterial effects such

healthy periodontal tissue without systemic disease, age

as root canal application and treatment of mucosal

54）（Ethical Review Committee of the Nippon Dental

damage in the mouth to accelerate wound healing by

University School of Life Dentistry at Niigata: approvalpromoting granulation of the wound. Moreover, gar-number ECNG-H-336）.

gling with chlorophyll compounds is useful for improv-

3 ．Human gingival fibroblasts

ing inflammation of periodontal tissue and halitosis in

Gingival tissue of the extracted teeth was washedpatients with gingivitis and periodontal disease throughwith phosphate buffer（phosphate buffered saline; Nissui

the antibacterial effect of chlorophyll2,6‒10）. Periodontal

Co., Tokyo, Japan）to remove impurities. The tissue wasdisease is an infectious disease caused by bacteria in

cut into about 1 mm3 pieces, and the tissue pieces were

the oral cavity. The oral bacteria cause inflammation ofplaced on a 35 mm culture dish. After adding a small

the periodontal tissue, which is thought to trigger theamount of Dulbecco’s Modified Eagle’s Medium

destruction of the periodontal ligament and resorption（DMEM）: Nutrient Mixture F-12 culture medium sup-of alveolar bone11）, and it has been reported that oral

plemented with 15％ fetal bovine serum（FBS）（JRbacteria are implicated in systemic diseases12‒14）. The

Scientific, Inc., CA, USA）
, 5μl/ml penicillin-streptomy-

prevalence of periodontal disease has been steadilycin（Invitrogen Corp., CA, USA）and 1μl/ml amphoter-increasing in recent years. A key to the treatment of

icin B（Invitrogen Corp.）（15％ FBS-containing DMEM）,

periodontal disease is to reduce dental plaque and peri-

the dish was incubated in a CO2 incubator at 37℃ in

odontopathic bacteria that can cause inflammation.

5％ CO2 and 95％ air for the primary culture. After

However, as the aging of the population progresses,

confirming the outgrowth from the tissue pieces, pri-

sufficient treatment for periodontal disease cannot bemary culturing was continued with exchanging theperformed in some cases due to association with sys-

medium every 2 days for about 2 weeks until thetemic diseases. Therefore, the importance of oral caremigrated cells reached confluence. The cells were thenand prevention of periodontal disease is widely recog-

cultured in 15％ FBS-containing DMEM and used fornized. Furthermore, drugs that reduce the adhesion of

the experiments. In addition, primary HGF purchased

plaque and periodontopathic bacteria and acceleratefrom the American Type Culture Collection（ATCC）

healing of periodontal tissue are being studied.

was cultured in DMEM containing 15％ FBS supple-

However, it remains largely unknown how SCCP acts

mented with 100-fold diluted penicillin streptomycin

to ameliorate inflammation and promote wound healing.

（FUJIFILM Wako Pure Chemical Corporation, Osaka,In this study, to elucidate the effects of SCCP on peri-

Japan）. Cell culture was performed under the sameDec, 2022

Effects of Sodium Copper Chlorophyllin on Human Gingival Fibroblasts in vitro

11

5.0×10 3 cells in 0.5％ FBS-containing DMEM andA

seeded in a 24-well plate（AGC TECHNO GLASS Co.,
Ltd., Shizuoka, Japan）. Twenty-four hours after seeding,the cells were exposed to SCCP at 10 nM and 100 nM
for 30 s. The medium was then replaced with 0.5％
FBS-containing DMEM, and the cells were cultured for
18 h16）. Total RNA of each cell was extracted using the
RNeasy mini Kit（Qiagen, PL, NLD）
. cDNA was synthesized using the High-Capacity RNA-to-DNA Kit. The

B

synthesized cDNA was then used for quantitative PCR.
TaqMan Gene Expression Master Mix（Thermo Fisher
Scientific Inc.）and TaqMan Gene Expression Assays（GAPDH: Hs02786624 gl, Cyclin D1: Hs0076553 ml）
were used for quantitative PCR and measured by a
Qubit3 Fluorometer（Thermo Fisher Scientific Inc.）.
From each data, relative quantitation was performed
using the comparative Ct method（comparative cycle
Fig. 1

Structure of sodium copper
chlorophyllin（A）and Cu（II）
chlorin e4 disodium salt（B）

17）
threshold; ΔΔCt）. GAPDH was used as an internal

control.
3 ）Statistics
The parametric test for comparing multiple unpaired
groups and the ANOVA test followed by the Tukey-

conditions.
4 ．Cell culture conditions

type test were used for the cell proliferation data andthe cyclin D expression data.

The cells（clinically isolated cell lines）were cultured
in 15％ FBS-containing DMEM. Cells were exposed to

Results

SCCP（Lot 60221）added to phosphate buffer at concentrations of 5 nM, 10 nM, 100 nM, 1μM and 10μM.
DMEM without SCCP was used as a control. Allantoin（10 nM）was used as a positive control.

1 ．Sodium copper chlorophyllin
The HPLC chromatogram of the SCCP（Lot 60221）isshown in Fig. 2（A）. The UV-visible absorption spec-

5 ．Measurements

trum of the largest peak（peak #10）was measured, and1 ）Cell proliferation

mass spectrum analysis was performed on peak #10.

Clinically isolated cells（Passage 3）were prepared at

The result is shown in Fig. 2（B）and（C）.

1.5×10 3 cells in 15％ FBS-containing DMEM and2 ．Cell proliferation

seeded in a 96-well plate（Becton Dickinson Biosciences,

On day 2 and 4 of culturing, no significant difference

CA, USA）. Twenty-four hours after seeding, the cells

was observed at any concentration compared to thewere exposed to SCCP at different concentrations.

control（Fig. 3）. On day 6 of culturing, HGF exposed to

After exposing for 30 s, the solution was replaced by

5 nM, 10 nM, 100 nM, 1μM SCCP and allantoin as posi-

15％ FBS-containing DMEM. The cells were exposed totive control showed a significant increase in cell prolif-

SCCP every 24 h. The cells were then counted every 48

eration compared to the control（Fig. 4）. On day 8 ofh for 8 days. Mitochondria reduction staining with Ala-

culturing, HGF exposed to 10 nM, 100 nM, and 1μM

mar Blue（Alamar Blue Cell Viability Reagent; Invitro-

SCCP showed significant levels of increase in cell prolif-

gen Corp.）was performed to measure cell proliferation

eration compared to the control. The plots of cell prolif-

with a fluorescence plate reader（Fluoroskan Ascenteration versus SCCP were bell-shaped with a peak at

FL; Thermo Fisher Scientific., MA, USA）.

100 nM（Fig. 5）.

2 ）Cyclin D1（Mitogen factor; cyclin D）

3 ．Mitogen factor: cyclin D

The cells purchased from ATCC were prepared at

HGF exposed to 100 nM SCCP expressed cyclin DODEP Vol. 2, No. 1

12

A

B

C

4,000

3,500

control
5nM
10nM
100nM
1µM
10µM
allantoin

3,500
3,000
2,500
2,000
1,500
1,000
500
0

1

2

4

6

8

day

Fig. 3 HGF Proliferation
Cell suspensions containing 1.5×103 cells with 15％
FBS-containing DMEM were implanted into a 96-wellplate. Twenty-four hours after seeding, the cells were
exposed to SCCP at different concentrations.

relative fluorescence intensity

relative fluorescence intensity

Fig. 2 Sodium copper chlorophyllin（A）HPLC chromatogram in peak #10 of SCCP（B）UV-visible absorption spectrum in peak #10 of SCCP（C）Mass spectrum in peak #10 of SCCP

3,000
2,500

＊
＊＊
＊＊

2,000

＊

＊＊＊
＊＊
＊＊

＊

＊

＊＊＊

1,500
1,000
500
0

control 5nM

10nM 100nM 1μM 10μM allantoin＊：p＜0.001＊＊：p＜0.01＊＊＊：p＜0.05

Fig. 4 HGF Proliferation day 6
Cell suspensions containing 1.5×103 cells with 15％
FBS-containing DMEM were implanted into a 96-wellplate. Twenty-four hours after seeding, the cells were
exposed to SCCP at different concentrations. The cells
were cultured for 6 days and measured（N＝6）.

Effects of Sodium Copper Chlorophyllin on Human Gingival Fibroblasts in vitro

relative fluorescence intensity

5,000

＊

4,500

＊＊＊

4,000
3,500

＊＊

＊

＊＊＊
＊

3,000

＊＊

Cyclin D（2-⊿⊿Ct）

Dec, 2022

＊＊

2,500
2,000
1,000

1.0
0.5
control

10nM

100nM
＊＊＊：p＜0.05

Fig. 6 Real-time PCR cyclin D1
The cells purchased from ATCC were prepared at 5.0×103 cells in 0.5％ FBS-containing

500
control 5nM

＊＊＊

1.5

0

1,500

0

2.0

13

10nM 100nM 1μM 10μM allantoin＊：p＜0.001＊＊：p＜0.01＊＊＊：p＜0.05

DMEM and seeded in a 24-well plate. Twenty-four hours after seeding, the cells were
exposed to SCCP at 10 nM and 100 nM for 30 s.

Fig. 5 HGF Proliferation day 8
Cell suspensions containing 1.5×103 cells with 15％

The medium was then replaced with 0.5％
FBS-containing DMEM, and the cells were cultured for 18 h. Cyclin D1 was measured byreal-time PCR（N＝6）.

FBS-containing DMEM were implanted into a 96-wellplate. Twenty-four hours after seeding, the cells were
exposed to SCCP at different concentrations. The cells
were cultured for 8 days and measured（N＝6）.

First, the ingredients of the SCCP were investigated.
mRNA at a significantly higher level compared to the

The UV-visible absorption spectrum of the largest peak

control（Fig. 6）.

#10 to the SCCP（Lot 60221）on the HPLC chromatogram was measured. As a result, maximum absorption
was observed at 405 and 627 nm（Fig. 2（B））. The

Discussion

absorption spectrum of the SCCP was very similar to
Periodontal disease is considered to be a chronic dis-that of Cu（Ⅱ）chlorin e4 disodium salt and Cu（Ⅱ）

ease caused by periodontal pathogens. Loss of adhesion

15）
chlorin e6 disodium salt（Fig. 1（B））
. Also, because

between the periodontal ligament and alveolar bone,

the sodium salt dissociates in the aqueous solution, theresorption of alveolar bone, and swelling of gingiva

mass spectrum of Cu（Ⅱ）chlorin e4 disodium salt

occur in the periodontal tissue that is affected by peri-（C33H34CuN4Na2O4）shows a mass-to-charge ratio of 614

odontal disease and associated inflammation11）. Because

in positive mode15）. This is consistent with Fig. 2（C）.

the prevalence of periodontal disease has increased, theThese results suggest that the main ingredient of theprevention of periodontal disease and suppression of

SCCP（Lot 60221）is Cu（Ⅱ）chlorin e4 disodium salt.

inflammation have attracted increasing attention in

We investigated the effect of the SCCP on cell prolif-

recent years.

eration. No significant difference was observed between

SCCP has various effects, including antiallergic, tis-days 2 and 4 of the culture as compared with the con-sue-activating, and deodorizing effects2,3）. In expectation

trol. HGF exposed to 5 nM, 10 nM, 100 nM, and 1μMof these effects, SCCP has started to be used in the field

SCCP showed significant levels of increase in cell prolif-of medicine. Particularly in the field of dentistry, SCCP

eration compared to the control on day 6 of culturing.

has been used for periodontal disease treatment and

Similar results were observed on day 8 of culturing,surgical treatment in expectation of its effects on sup-showing a bell-shaped concentration-proliferation curve

7‒10）

.

with a peak at 100 nM. This result shows the optimum

However, although there are many clinical reports,

concentration of SCCP for HGF in this experiment. 10

there are few basic research reports on the use of

μM SCCP showed no significant difference in cell pro-

SCCP. In this study, we investigated the changes in theliferation compared to untreated controls. In this study,

properties of HGF in vitro to elucidate physiological

we did not evaluate the cytotoxicity of SCCP by mea-changes in periodontal tissue exposed to SCCP.

suring the lactate dehydrogenase activity, mitotoxicity

pressing halitosis and promoting wound healing

ODEP Vol. 2, No. 1

14

assays, apoptosis assay, etc. However, 10μM of SCCP

cyclin D1 expression, proliferation, and migration of

did not show a lower value in cell proliferation com-

HGF exposed to SCCP. In this study, the exposure time

pared to the untreated control. Therefore, it was con-

was set to 30 seconds, assuming that SCCP was usedsidered that high concentrations of SCCP had no cyto-during gargling. The results suggest that SCCP istoxic effect. This result showed the optimum concentra-

appropriate for short-term exposure such as toothtion of SCCP for HGF in this experiment. In this study,

brushing and gargling. These findings suggest thatallantoin, whose cell proliferation effect and wound

exposure of periodontal tissue to SCCP induces cyclin

healing effect by cell migration were already confirmed,

D1 expression and promotes proliferation and migration

18,19）. In this study, SCCP

of gingival fibroblasts. Therefore, SCCP could be effec-

showed cell proliferation effect equal to or higher thantive in promoting wound healing in periodontal tissue

that of allantoin. The mitogen factor, cyclin D1, is aand contribute to the prevention of periodontal disease.

major regulator of cell cycle and cell proliferation mark-Future studies should clarify the mechanism by which

ers20）. In this study of cyclin D1, 100 nM SCCP signifi-

SCCP promotes cyclin D1 expression.

was used as a positive control

cantly promoted cell proliferation of HGF, so 100 nM
SCCP and a low concentration of 10 nM SCCP wereConclusions

used as concentration conditions. In addition, since theprimary HGF is not affected by clinical conditions, it

The results of this study suggest that SCCP is appro-

was considered to be suitable for examining gene

priate for short-term exposure such as toothbrushingexpression and was used. This study showed a signifi-

and gargling. The findings suggest that exposure ofcantly increased level of cyclin D1 expression in HGF

periodontal tissue to SCCP induces cyclin D1 expres-exposed to 100 nM SCCP. Although no significant dif-sion and promotes proliferation and migration of gingi-

ference was observed between 10 nM SCCP as com-

val fibroblasts. Therefore, SCCP could be effective in

pared with the control, it is considered that continued

promoting wound healing in periodontal tissue and con-

exposure of SCCP to HGF increased the expression oftribute to the prevention of periodontal disease. Future

cyclin D1 and promoted cell proliferation. Lee DH et al.

studies are needed to clarify the mechanism by which

reported that recombinant growth factor mixtures

SCCP promotes cyclin D1 expression.

（RGFM）significantly increased cyclin D1 expression .

20）

As a result, it was revealed that wound closure in mice

Conflict of Interest

was accelerated, re-epithelialization was promoted, andThe authors declare no conflicts of interest associatedwith this manuscript.

inflammatory cell infiltration was reduced20）. Furthermore, Zhiping et al. reported increased adhesion anddecreased motility of cyclin D1-deficient mouse embryonic fibroblasts compared to wild-type mouse embryonic fibroblasts21）. These reports suggest that increased
cyclin D1 expression promotes proliferation and migration of cells involved in tissue repair20,21）. Furthermore,
it has been reported that enamel matrix derivative andfibroblast growth factor, which have an action of promoting periodontal tissue repair, induce human gingival
fibroblasts into the S phase of the cell cycle and promote cell proliferation22‒25）. In the present study, exposure to SCCP promoted proliferation of HGF in a concentration-dependent manner up to 100 nM. This result
suggests that SCCP promotes cell activity. Recent studies have revealed that the increased cyclin D1 expression promotes the proliferation and activity of cells.
This study also showed significantly increased levels ofReferences
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15）Simaremare ES, Djalin AD, Tjahjono DH. Photophysicaland physicochemical properties of Cu（Ⅱ）chlorin e4
and Cu（Ⅱ）chlorin e6 as a lead compound of photosensitizer for PDT. Indonesian J Pharm 2015; 26: 29‒36.
16）Reiri T. The effect of basic fibroblast growth factor oncell cycle in human gingival fibroblasts from nifedipine
responder and non-responder. J Oral Sci 2004; 46: 37‒44.
17）Livak KJ, Schmittgen TD. Analysis of relative gene18）Cahen R, Pessonnier A. Pharmacological study of dihydroxyaluminum allantoinate and chlorhydroxyaluminum
allantoinate. Ⅲ. Anti-ulcerous effect. Ann Pharm Fr
1962; 20: 704‒713.
19）Cahen R, Pessonnier A. Pharmacological study of dihydroxyaluminum allantoinate and chlorhydroxyaluminum
allantoinate. Ⅳ. Effect on experimental drug-induced
ulcer. Ann Pharm Fr 1963; 21: 215‒222.
20）Lee DH, Choi KH, Cho JW, Kim SY, Kwon TR, Choi SY,
Choi YM, Lee J, Yoon HS, Kim BJ. Recombinant growth
factor mixtures induce cell cycle progression and theupregulation of type Ⅰ collagen in human skin fibroblasts, resulting in the acceleration of wound healing
processes. Int J Mol Med 2014; 33: 1147‒1152.
21）Zhiping L, Chenguang W, Xuanmao J, Yinan L, Maofu F,
Andrew AQ, Chip D, Jianguo Y, Maozheng D, Xiaoming
J, Xueping Z, Anping L, Peter B. Cyclin D1 regulates
cellular migration through the inhibition of thrombospondin 1 and ROCK signaling. Mol Cell Biol 2006; 26:4240‒4256.
22）Narges A, Payam GD, Morteza HC, Mahbobe GA, Abbas
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23）Zeldich E, Koren R, Nemcovsky C, Weinreb M. Enamel
matrix derivative stimulates human gingival fibroblast
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24）Wyganowska-Swiatkowska M Urbaniak P, Lipinski D,
Szalata M, Kotwicka M. Human gingival fibroblast
response to enamel matrix derivative, porcine recombinant 21.3-kDa amelogenin and 5.3-kDa tyrosine-richamelogenin peptide. Hum Cell 2017; 30: 181‒191.
25）Chen L, Lei Z, Wang L. In vitro experimental research ofbasic fibroblast growth factor/chitosan/polylactic acidscaffolds in periodontal tissue regeneration. Chinese J
Tissue Eng Res 2017; 21: 4106‒4112.

402‒408.

ODEP Vol. 2, No. 1

16

Original Article

ODEP 2（1）：16～24，2022

Three-year Retrospective Follow-up of Cases of Root Canal Obturation
with a Bioactive Glass-based Root Canal Sealer
Ayako WASHIO, Hiroki MIURA, Miki SUEMATSU,
Kazumasa MURATA, Ryosuke AIHARA, Masataka FUJIMOTO,
Ai ORIMOTO and Chiaki KITAMURA
Division of Endodontics and Restorative Dentistry, Department of Oral Functions, Kyushu Dental University

Abstract
Purpose: Bioceramics-based root canal sealers are becoming essential in modern endodontics worldwide
and are currently being used in Japan; however, there have been no follow-up studies on teeth treated using
these sealers. The purpose of this study was to retrospectively evaluate 3-year follow-up data pertaining toteeth treated with a bioceramics-based root canal sealer.
Methods: Patients treated at the Division of Tooth Therapy（Endodontics and Restorative Dentistry）ofKyushu Dental University Hospital and a private dental clinic were recruited, and 127 teeth that were obturated with a bioactive glass-based root canal sealer were followed up for 3 years. The rates and incidence offunctionally surviving and extracted teeth were analyzed. Additionally, 56 teeth with 3-year follow-up radiographs were assessed using the periapical index（PAI）score.
Results: One hundred and fifteen teeth（91％）survived functionally, and 12 teeth（9％）were extracted
during the 3 years. The 12 extracted teeth were initially diagnosed as non-preservable by the dentist
because of root cracks or fractures and extensive bone defects, but were endodontically treated in accordance with the patients’ strong requests. There were significant associations between functional surviving
and extracted teeth and tooth type, subjective symptoms, initial treatment/re-treatment, and cracks/fractures. In contrast, there was no significant difference between surviving and extracted teeth according to theroot canal obturation technique, pain immediately after root canal obturation, and overfilling of the root canal.
In the comparison according to PAI scores, the rates of scores 1 and 2 increased, whereas those of scores 3
and 4 decreased.
Conclusion: This 3-year retrospective follow-up study of root canal-obturated cases revealed that the bioactive glass-based root canal sealer evaluated herein, a bioceramics-based sealer, can be applied to achievefavorable outcomes in endodontic therapy.
Key words: bioactive glass, root canal sealer, root canal obturation, follow-up

Corresponding author: Ayako WASHIO, Division of Endodontics and Restorative Dentistry, Department of Oral Functions,
Kyushu Dental University, 2‒6‒1, Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803‒8580, Japan
TEL: ＋81‒93‒285‒3082, FAX: ＋81‒93‒581‒5399, E-mail: r05washio@fa.kyu-dent.ac.jp
Received for Publication: August 18, 2022/Accepted for Publication: September 27, 2022
DOI: 10.11471/odep.2022‒003

Dec, 2022

Follow-up of Root Canal Obturation with a Bioceramics-based Root Canal Sealer

17

to this study. Ethical approval for the study was
obtained from the ethics committee of Kyushu Dental

Introduction

University（approval no: 17‒39; Kyushu Fukuoka,
Successful endodontic therapy depends on adequate

Japan）. One hundred and twenty-seven teeth that weremechanical shaping, a copious and effective irrigation

endodontically treated by six dentists（two endodontists

protocol, and 3-dimensional obturation of the root canaland four dentists with at least 5 years of experience）

1）

system . In a previous study, the association between

and then followed up for 3 years were selected for thisfailure of root canal treatment and poor root canal obtu-

study.

ration was demonstrated

2,3）. The invasion of microor-

2 ．Endodontic treatment procedures

ganisms into the interfacial region between the root

Individual dentists examined and diagnosed eachcanal dentinal wall and filling materials needs to bepatient, and the same dentist was assigned for endodon-

prevented to avoid re-infection

4‒6）. Achieving a propertic treatment. Local anesthesia was administered forseal depends on the ability of the filling material to bindpulpectomy. The anesthetic effect was verified based on

to the dentinal wall and on the root canal obturation

subjective symptoms（tingling and numbness）. In casetechnique. These findings highlight the importance ofof severe loss of tooth structure, the pre-endodontic

root canal sealers. Consequently, preventing microleak-build-up of the crown was prepared to facilitate rubber

age by ensuring proper root canal obturation is essen-

dam placement and temporization of the tooth between

tial to obtain lasting clinical success.

visits.

Bioceramics-based endodontic biomaterials have

After isolation of the tooth by a rubber dam, thegained popularity in modern endodontics because ofaccess cavity was prepared using sterile diamond

their physicochemical properties and biocompatibility,

points and carbide burs. The canal patency was

which is the ability of a material to achieve a stable andchecked using a #10 K-file（MANI Inc., Tochigi, Japan）,

7,8）. In particu-

and a glide path was established using a #15 K-filelar, bioceramics-based root canal sealers have been

（MANI Inc.）. The working length was determined

incorporated into endodontic procedures to improve theusing an electronic apex locator（Root ZX Ⅱ; Morita,

outcome of root canal treatment9）. Several studies also

Kyoto, Japan）. In cases where a reliable electronic apex

indicated that bioceramics-based root canal sealers usedlocator reading could not be obtained, a radiograph waswith a single-cone technique are useful for root canaltaken to confirm the working length. The canals were

obturation and that teeth obturated using these sealers

irrigated with 3％ sodium hypochlorite（NaClO; Nippon

confer a favorable outcome10,11）.

Shika Yakuhin Co., Ltd.）using an endodontic syringe

advantageous host response when appliedThough bioceramics-based root canal sealers arewith a closed-ended side-vented needle after each filing.

used in Japan, there have been no reports on the fol-

The master apical file size was case-specific and deter-

low-up of teeth obturated using these sealers. The pur-mined based on the initial canal size. In re-treatment

pose of this study was to retrospectively evaluate fol-cases, the previously applied root canal filling materials

low-up data pertaining to non-surgical root canal treat-

were removed using a combination of ultrasonics, ament using Nishika Canal Sealer BG（CS-BG; Nippon

gutta percha solvent（GP-Solvent; Nippon Shika Yakuhin

Shika Yakuhin Co., Ltd., Yamaguchi, Japan）, an easilyCo., Ltd.）, rotary instruments, and K-files.

available and widely used bioceramics-based root canalsealer.

After root canal preparation, all the canals were irrigated by applying ultrasonic activation（P-Max plus;
Acteon Inc., France）at two sets of 30 s each with 3％

Materials and Methods

NaClO and 3％ ethylenediaminetetraacetic acid
（EDTA）solution（Nippon Shika Yakuhin Co., Ltd.）.

1 ．Patient selection

Subsequently, the canals were washed with sterilized

Patients treated at the Division of Tooth Therapy

saline（Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan）.

（Endodontics and Restorative Dentistry）of Kyushu

After the completion of irrigation, the canals were

Dental University Hospital and a private practice clinic

dried using an endo aspirator and paper points and（Miura Dental Clinic, Fukuoka, Japan）were recruited

filled with calcium hydroxide paste（Calcipex Ⅱ; Nippon

ODEP Vol. 2, No. 1

18

Shika Yakuhin Co., Ltd.）. The coronal cavity was sealed

3-year follow-up period was archived for 56 of the 115

with an intermediate restorative material.

surviving teeth, and the periapical region status at each

After improvement of the patient’s symptoms, thetime-point―pre-operative（Pre-op）, root canal obtura-root canals were obturated with CS-BG and gutta per-tion（RCF）, and 3-year follow-up―were assessedcha points（GP; Morita）. CS-BG is a two-phase paste that

according to a periapical index（PAI）score12,13）by two

includes Paste A, which consists of fatty acid, bismuth

blinded, independent, and trained examiners, as follows

subcarbonate, and silicon dioxide, and Paste B, which（Figure 1）:

consists of magnesium oxide, calcium silicate glass（atype of bioactive glass）, and silicon dioxide. CS-BG was
prepared according to the manufacturer’s instructions.
After irrigation and drying of the canals with root canal

PAI 1: Normal periapical structure
PAI 2: Bone structural changes indicating, but notpathognomonic for, apical periodontitis
PAI 3: Bone structural changes with some mineral

vacuum and paper points, a master GP was adapted,

loss characteristic of apical periodontitis

and the canals were obturated using the single-cone

PAI 4: Well-deﬁned apical radiolucency

technique（Single）, multi-cone technique without condensation（Multi-Non-press）, or lateral condensation
technique（Multi-Lateral）, as follows:
・Single: A dentist used a single master GP and root
canal sealer.

PAI 5: Radiolucency with radiating expansion of bone
structural changes
Multi-rooted teeth were assigned the highest score
for any of the roots.
4 ．Statistical analysis

・Multi-Non-press: A dentist used a single master GP,

The data were analyzed using the chi-squared test;

several accessory GPs, and a root canal sealer,

SPSS Statistics 25.0 software（IBM Corp., Armonk, NY,

without lateral condensation.

USA）was used. The statistical significance was set at

・Multi-Lateral: A dentist used a single master GP,

p＜0.05.

several accessory GPs, and a root canal sealer, withcondensation.

Results

Excess sealer and GPs were removed using a heatedplugger. The coronal cavity was sealed with an inter-

The patient demographic data are shown in Figure 2.

mediate restorative material. The obturation was veri-

A total of 127 teeth were included in this study. Thir-

fied using radiography. Permanent restorations of thety-eight（30％）teeth belonged to male patients, and 89

teeth were performed as soon as possible.

（70％）to female patients（Figure 2-a）. The patient ages

3 ．Follow-up and outcome assessment

ranged between 10 and 89 years as follows（age range,

Patient attributes including gender, age, arch, type of

number of teeth）: 10‒19, 1（1％）; 20‒29, 2（2％）; 30‒39,

tooth, subjective and objective symptoms, initial treat-5（4％）; 40‒49, 15（12％）; 50‒59, 25（20％）; 60‒69, 31

ment or re-treatment, endodontic treatments（pulpec-（24％）; 70‒79, 39（31％）; and 80‒89, 9（7％）teeth（Fig-tomy or infected root canal treatment）, detection ofure 2-b）. Figure 2-c shows the arch and tooth types.

root crack（Crack）or root fracture（Fracture）, rootSeventy-five（59％）teeth were in the maxilla, and 52

canal obturation techniques（Single, Multi-Non-press, or（41％）in the mandible. Forty-eight（38％）of the treatedMulti-Lateral）, presence or absence of pain immediately

teeth were located in the anterior segment, 29（23％）

after root canal obturation, presence or absence ofwere premolars, and 50（39％）were molars. Thirty-four

overfilling of the root canal at the time of filling, and（27％）teeth underwent initial treatment and 93（73％）

functionally surviving teeth（Survived）or extracted

underwent re-treatment（Figure 2-d）. Figure 2-e showsteeth（Extracted）at 3 years were examined. Informa-

the incidence rates of“Survived”and“Extracted”at 3tion on the extracted teeth was sought from the patient

years. One hundred and fifteen（91％）teeth survived

or the referring dentist, or was derived from thefunctionally, and 12（9％）teeth were extracted withinpatient’s records. In addition, the incidence of Survived

three years. Of the 115 teeth that survived, 108 wereor Extracted was examined. Clinical follow-up was per-diagnosed as preservable and seven as non-preservable

formed at 3 years for each tooth.

at the time of the initial examination; however, thePeriodical analysis of radiographic images during theteeth were treated endodontically in accordance with

Dec, 2022

Follow-up of Root Canal Obturation with a Bioceramics-based Root Canal Sealer

19

Table 2 shows the incidence of root canal obturationin the Survived and Extracted groups. There were no
significant associations between Survived/Extracted
and the root canal obturation technique, pain immediately after root canal obturation, and overfilling of theroot canal immediately after the obturation.
Figure 3 shows the PAI score of the 56 surviving
teeth with 3-year follow-up radiographs, and the mean/
median/mode at Pre-op, RCF, and 3-year follow-up.
ab

During the 3-year follow-up period, the rates of PAIscores 1 and 2 increased in a time-dependent manner,
whereas those of scores 3 and 4 decreased.

Discussion
In this study, we retrospectively evaluated follow-up
data pertaining to non-surgical root canal treatmentusing CS-BG, an easily available and widely used bioactive glass-based root canal sealer. CS-BG was developedin 2017 and is now being applied as a bioactive glasscdbased root canal sealer and pulp-capping material,
Nishika Canal Sealer BG multi（Nippon Shika YakuhinCo., Ltd.）. CS-BG is delivered as two pastes, which canbe mixed as needed; this promotes consistency and efficiency in formulating and using this biomaterial in aclinical setting. The superior flowability and the ability
to slightly expand upon setting allow this sealer to be
used with the single-cone technique14‒17）. Several in
vitro and in vivo studies have also reported that CS-BG
has excellent biocompatibility with periapical tissues15‒17）. Additionally, a clinical study reported thateFig. 1

Examples of teeth with periapical index（PAI）
score of 1（a）, 2（b）, 3（c）, 4（d）, and 5（e）

CS-BG can reduce the distress of patients during root
canal obturation18）. However, the clinical significance of
these characteristics remains unclear.
In the present study, 12 extracted teeth that had acracked or fractured root were endodontically treatedthe patients’ strong requests（data not shown）. All thein accordance with the patients’ preferences, even

12 extracted teeth were diagnosed as non-preservable

though they were diagnosed as non-preservable at theat the time of the initial examination, but were end-

time of the initial examination. Additionally, many of theodontically treated in accordance with the patients’

extracted teeth had thinning of root dentin pre-opera-

strong requests（data not shown）.

tively or post-operatively（data not shown）. It has been

Table 1 shows the incidence of the various patientreported that thinning of root dentin by not only end-

characteristics and operative characteristics in the Sur-odontic treatment but also the length and thickness of

vived and Extracted groups. There were significant

the post hole affects root cracking and fracture19）. A

associations between Survived/Extracted and type ofprevious study including Kaplan-Meier analysis of

tooth, subjective symptoms, initial treatment/re-treat-

5-year follow-up data reported that most lost teeth arement, and Crack/Fracture.

extracted within 2 years after treatment20,21）. ConsistentODEP Vol. 2, No. 1

20
（a）
70％

30％

Male
Female
100％

0％

（b）
1％

2％

4％

12％

20％

31％

7％

10－19 years
20－29 years
30－39 years
40－49 years

24％

50－59 years
60－69 years

0％

（c）

100％

28％

10％
11％

80－89 years

Maxillary anterior

12％

Maxillary premolor
Maxillary molor

19％

20％

70－79 years

Mandibular anterior
0％

100％

Mandibular premolor
Mandibular molor

（d）
27％

73％

0％

Initial treatment
100％

Re-treatment

（e）
91％
0％
Fig. 2

9％100％

Survived
Extracted

Demographic data（a）gender,（b）age,（c）arch and type of tooth,（d）initial treatment or re-treatment,（e）teeth that survived or were extractedwith the literature, in this study, 11 of the 12 teeth

with root cracks or fractures was 5 and 3, respectively

were extracted within 2 years of treatment（data not（data not shown）. Therefore, teeth with root cracks orshown）. Interestingly, seven functionally surviving

fractures can survive and retain function in the absence

teeth with root cracks or fractures were diagnosed asof extensive bone defects.

non-preservable at the initial examination but survived

Previous studies have examined the incidence of sur-and functioned after endodontic treatment. Based onvival or extraction in root canal-obturated teeth andthe evaluation of the PAI score, the median score of thereported that the survival of such teeth was signifi-

12 extracted teeth and 7 functionally surviving teeth

cantly related to patient age 22）. Also, it has beenDec, 2022

Follow-up of Root Canal Obturation with a Bioceramics-based Root Canal Sealer
Table 1

21

Patient characteristics and pre-operative variables of teeth that survived（Survived）or were extracted（Extracted）
Total, n＝127

GenderMale

AgeFemale＜50 years≥50 years

Survived

Extracted

35
80

3
9

.696

21

2

.891

94

10

（n＝115） （n＝12）

Arch

Maxillary

69

6

46

6

Type of tooth

Mandibular
Anterior

47

1

Premolar
Molar

29

0

39
70

11
0

Subjective symptoms

No symptoms
Symptoms
No symptoms

45

12

Objective symptoms

372

Symptoms
Initial treatment

78

10

Initial-/Re-treatment

34

0

81

12

Re-treatment
Endodontic treatments Pulpectomy
Infected root
canal treatment
Absence
Presence

Crack/Fracture

22

0

93

12

108
7

7
5

p value.503.0004＊.0001＊
.268
.028＊
.096

.0001＊＊：χ2-test（p＜.05）.

Table 2

Root canal obturation-related variables of teeth that survived（Survived）and wereextracted（Extracted）
Survived Extracted（n＝115） （n＝12）

Total, n＝127
Root canal obturation technique

Pain immediately after root canal obturation
Overfilling of root canalSingleMulti-Non-press
Multi-LateralNo pain
Pain
Absence
Presence

34
76
5
113
2
77
38

4
7
1
11
1
10
2

p value
.772

.152
.245

reported that the pulp chamber is often constricted intreatment29,30）. However, the relationship between thepatients over 50 years of age, making the procedure

presence or absence of subjective symptoms and fol-

more complicated and affecting the prognosis 23‒25）.

low-up has rarely been reported previously, although

However, the present study showed that age did not

the presence of subjective symptoms is believed to beaffect the survival of root-obturated teeth; the reason

important in the need for root canal treatment. The

for this difference in results remains unclear.

results of the present study suggest that a molar tooth

The most common reason for tooth extraction after
endodontic treatment is tooth fracture

26,27）, and molars

are more likely to be extracted after endodontic treat28）

with subjective symptoms and crack or fracture is
likely to be extracted within 3 years if the tooth isre-treated.

ment than other teeth . In addition, re-treatment has

Root canal obturation techniques have been reported

been reported to have a lower success rate than initial

to be related to the extrusion of the root canal sealer

ODEP Vol. 2, No. 1

2256

sealers, it is important to select materials with satisfactory physicochemical properties and low toxicity43）. In
Score 5

Teeth

Score 4
Score 3
Score 2
Score 10

the present study, there were no significant associations between the 3-year follow-up data and apically
extruded sealers in the periradicular tissues at RCF.
The apically extruded sealers of several teeth wereabsent in the periradicular tissues on 3-year follow-up
radiographs, and a collateral（or original canal）seen in
the root canal at pretreatment appeared to have been

Pre-opRCF

3-year

Mean

3.2

3.0

2.5

Median

3

3

2

Mode

3

3

2

sealed by the obturation material（data not shown）.
CS-BG has been reported to be biocompatible with periapical tissues38,39）, and a case report showed that thepresence of CS-BG in periapical tissues did not affectthe healing of periapical lesions44）. Therefore, the sin-

Fig. 3 Periapical index（PAI）score distribution,
mean, median, and mode at the pre-operative stage（Pre-op）, root canal obturation（RCF）, and 3-year follow-up

gle-cone technique using CS-BG is expected to be useful. However, the intentional and excessive extrusion of
the sealer into the periapical region should be avoided.
The PAI score12,13）was employed in the current analysis. This scoring system has been used in many longitudinal studies, and its significant prognostic value haswith post-obturated pain, but these factors do not affect31,32）

been proven, particularly in repeated radiological evalu-. The

ations. Many authors have suggested that the time

results of the present study are in agreement withperiod for evaluating healing parameters in teeth withthose of these previous studies. An ideal root canal

periapical lesions is 4 to 5 years45,46）. However, it was

obturation should ensure that the filling materials reachdemonstrated that a 1-year period may be ideal to con-the cement-dentinal junction33）. Studies have shown thattrol for periapical changes in bone density when using

the highest success rate of endodontic treatment is

PAI scores47）. The PAI scores were divided into healed

observed in teeth with root canal fillings ending 0‒2（PAI≤2, no signs or symptoms）and non-healed（PAI

mm short of the radiographic apex, and cases with

＞2, with/without signs or symptoms）7,48）. The PAI

underfillings or overfillings are associated with signifi-

scores for 56 of the 115 surviving teeth included in thisthe outcomes of endodontically treated teeth

cantly lower success rates

33‒35）. In contrast, uninten-

study suggest that root canal obturation using a bioac-

tional extrusion of the root canal sealer is a common

tive glass-based root canal sealer is acceptable for

event that, in low amounts, is usually well tolerated byachieving favorable endodontic outcomes. However, thethe periradicular tissues36,37）. Evidence indicates that

present data are insufficient because of the small sam-the unintentional apical extrusion of root canal fillings

ple size; follow-up studies for longer periods are cur-seems to have no direct correlation with treatment fail-rently underway.

ure, provided that infection is absent38‒41）. The unintentional extrusion of filling materials can occur owing toConclusions

the lack of apical constriction as a result of inflammatory apical root resorption or an incompletely formed

The findings of this retrospective study suggest thatroot apex, or over-instrumentation due to errors inbioactive glass-based root canal sealers are useful forworking length assessment42）. In other words, the inci-

cases diagnosed as preservable at the start of endodon-

dence of sealer extrusion seems to be related more

tic therapy, and that tooth obturation by the single-coneclosely with operator parameters and root canal prepa-

technique using this sealer is a viable option. Addition-

ration and filling techniques than with the root canalally, the results show that in the present study, the rea-sealer selected for use. However, to avoid accidents andson for teeth extraction in most cases was not underly-complications caused by the extrusion of root canaling periapical disease but root cracks or fractures with

Dec, 2022

Follow-up of Root Canal Obturation with a Bioceramics-based Root Canal Sealer

extensive bone defects detected at the time of the initial examination.

23

apical periodontitis. Endod Dent Traumatol 1986; 2: 20‒34.
13）Orstavik D. Time-course and risk analyses of the devel-

Conflict of Interest
The authors declare no conflicts of interest associatedwith this manuscript.

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Dec, 2022

25

Original Article

ODEP 2（1）：25～32，2022

Influence of Light Irradiation Modes on the Polymerizationof Light-curing Resins in a Root Canal Model
Kazumasa MURATA, Hiroki MIURA, Hiroshi IKEDA1,
Shinji YOSHII2, Ayako WASHIO and Chiaki KITAMURA
Division of Endodontics and Restorative Dentistry, Department of Oral Functions, Kyushu Dental University
1

Division of Biomaterials, Department of Oral Functions, Kyushu Dental University

2

Department of Promoting Learning Design Education, Department of Health Promotion, Kyushu Dental University

Abstract
Purpose: Fiber posts with resin cores have been widely used as direct build-up materials for the restoration of endodontically treated teeth. However, the influence of light irradiation mode, including the irradiation intensity and time, on resin polymerization in the apical root canal remains an open research problem.
This in vitro study investigated the influence of light irradiation mode on the polymerization of different
light-curing resins with and without a fiber post.
Methods: A translucent fiber post, namely i-TFC Luminous fiber, was used in combination with five commercial light-curing resins: i-TFC System Universal（UN）, i-TFC System Blue（BL）, i-TFC Luminous core LC
flow（LC）, Filtek U（FU）, and MI core LC flow（MI）. Each resin was filled into an artificial root canal model（diameter: 3 mm, depth: 15 mm）with or without the fiber and light-cured using a handy light-curing unit in
two irradiation modes: mode-F3: 2,000 mW/cm2 for 3 s, and mode-10: 1,200 mW/cm2 for 10 s×3. The cured
resins were characterized by their polymerization ratios, depths, and optical transmittances.
Results: The polymerization ratio of each resin was significantly higher in mode-10 than in mode-F3 withand without the fiber post. In the fiber-post groups with mode-10, the polymerization ratio was over 96％ foreach resin. In the fiber-post groups with mode-F3, the polymerization ratio of each resin was relatively low:90.5±3.6 for UN, 92.8±4.8 for BL, 85.2±3.7 for LC, 92.7±2.3 for FU, and 75.1±5.2 for MI. The polymerization
depth of each resin with the fiber post reached 15 mm for each mode. The LC resin produced the highest
optical transmittance, followed by the UN, BL, MI, and FU resins.
Conclusion: Longer light irradiation and the use of a fiber post were found to be essential to achieve sufficient resin polymerization in the apical root canal.
Key words: fiber, polymerization, root canalCorresponding author: Hiroshi IKEDA, Division of Biomaterials, Department of Oral Functions, Kyushu Dental University,2‒6‒1, Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803‒8580, Japan
TEL: ＋81‒93‒582‒1131, FAX: ＋81‒93‒592‒1699, E-mail: r16ikeda@fa.kyu-dent.ac.jp
Received for Publication: September 14, 2022/Accepted for Publication: October 24, 2022
Doi: 10.11471/odep.2022‒004

ODEP Vol. 2, No. 1

26

product.

Introduction
Materials and Methods
Fiber-post/resin-core construction systems havebecome more popular for preparing tooth abutments

1than conventional metal post systems because their

Table 1 lists the fibers and resins used in the fiber-mechanical properties, such as the elastic modulus, arepost/resin-core system. The fiber was used at thesimilar to those of dentin1‒3）. In the direct restoration of

as-received length（18 mm）without cutting to minimize

the tooth in a system, the resin is filled into the tooth

light scattering at the cutting interface.

root canal with a fiber, followed by polymerization via

2

light irradiation. The degree of polymerization of theMaterials

Polymerization ratio for the fiber-post/resincore system

resin is one of the most important factors influencing

The polymerization weight of the resin core was

the mechanical and physicochemical properties of theevaluated by weight changes before and after light irra-material4‒6）, and it affects the long-term success of

diation according to a previous study19）. A Teflon block

tooth restoration

7, 8）. To maximize the performance ofwith a cylindrical cavity（diameter: 3 mm, depth: 15

the fiber-post/resin-core construction system, it is pref-mm）was fabricated and used as the artificial root canalerable to polymerize the resin as much as possible.

model（Fig. 1-a）. Figure 1-b shows the light polymeriza-

However, complete polymerization of the resin in antion process of the resin. Prior to the test, the Teflon

apical root canal is difficult because of high humidity

mold, shielding silicone, and acrylic plate were weighed

and light shielding in the narrow cavity（A）. The fiber post was also weighed（B）. Resin was

9, 10）.

To overcome this issue, much effort has been madeinjected into the cavity, and the fiber was inserted intoon optimizing the polymerization of resin in the root

the cavity through an acrylic plate. Subsequently, thecanal. For instance, a study reported that the transpar-surrounding mold, except for the top surface, was cov-

ency of the fiber post effectively influences the polym-

ered with silicone to shield it from ambient light. This11）

erization of the resin . The polymerization of the resin

setup imitates a dentin root canal, in which irradiationin an apical root canal was higher with translucent

light can enter the root canal cavity from the coronal

12）

fiber posts than with an opaque fiber post . The fiber-

direction. Light irradiation was carried out at a distancepost type and dowel space levels influence resin polym-of 3 mm from the top surface of the root canal using a13）erization . Light polymerization of the resin was insuf-

handheld light-curing unit（G-Light Prima, GC, Tokyo,

ficient in areas deeper than 4‒6 mm and further away

Japan）in two different modes: mode-F3, 2,000 mW/cm2

from the fiber post14）. The degree of conversion of thefor 3 s; and mode-10, 1,200 mW/cm2 for 10 s×3. After

resin decreased as the post length increased

15, 16）. Insuf-light irradiation, the total weights of the mold, silicone,

ficient polymerization of the resins was observed inplate, resin, and fiber were measured（C）. The resultant

deep areas of the root canal using dual-curing res-resin was removed from the mold and washed by

17, 18）. In addition, our previous study demonstrated

immersing in acetone for 30 s to eliminate the uncuredthat the combination of a highly translucent fiber post

resin. The washed resin was kept under ambient condi-

and light-curing resin facilitates resin polymerization intions at 25℃ for 24 h to dry, and then weighed（D）. Theins

deeper areas of the root canal

19）. However, there isroom for further investigation on the influence of lightpolymerization ratio of the resin was determined using
the equation:

irradiation conditions（intensity and exposure time）on

Polymerization ratio（％）

resin polymerization in the root canal.

＝（D－B）/（C－B－A）
×100

This study investigated the influence of light irradia-……（1）tion mode on the polymerization of different light-cur-Using this procedure, the polymerization ratio of eaching resins in a fiber-post/resin-core construction sys-resin was measured with and without the fiber（n＝5）.

tem. An in vitro experiment was performed using an
artificial root canal model with five commercial
light-curing resin products and one commercial fiber

3

Polymerization depth for the fiber-post/resincore system

After the polymerization ratio test, the resultant res-Dec, 2022

Polymerization of Light-curing Resins in a Root Canal Model
Table 1

27

Materials used in this study

Product name

Manufacturer

Code

Fiber post

i-TFC Luminous fiber

Sun Medical Corp., Moriyama, Japan

Light curing resin

i-TFC Luminous core LC flow
i-TFC System Universal
i-TFC System Blue
Filtek U
MI core LC flow

Sun Medical Corp., Moriyama, JapanSun Medical Corp., Moriyama, JapanSun Medical Corp., Moriyama, Japan
3M Corp., MN, USA
GC Corp., Tokyo, Japan（⌀1.0 mm/tapered）

LC
UN
BL
FU
MI

（c）

（a）
3.0 mm

Acrylic plate

Teflon block
Measured value＝Polymerization depth

15.0
20.0 mm
mm

10.0
mm

Teflon block

Silicone（b）
Fiber post（B）

Acrylic plateCore resin

Root canal post
cavity model（A）
Light-curing
unit

Filling core resin

Mode-F3orMode-10

18 mm

Light
irradiation（C）

Isolatedfrom model

Inserting fiber post

Immersed into
acetone to remove
unpolymerizedresin for 30 s andthen washed anddried for 24 h（D）

Fig. 1 Experimental system for polymerization ratio and depth measurement（a）Image of a Teflon mold with shielding silicone for the root canal model.（b）Procedure for measuring polymerization
ratio and depth of the resin in the root canal.（c）Image of cured resin after acetone washing.

ins were used for polymerization depth measurements,

each resin with and without the fiber（n＝5）.

as shown in Fig. 1-c. The length of the resin was mea-

4

sured using a digital scale to determine the polymeriza-

The transmission spectra of the resin were measured

tion depth. Depth measurements were performed forin the wavelength range of 200‒800 nm using an ultra-

Transmittance of resin

ODEP Vol. 2, No. 1

28

Fig. 2

Representative images of polymerization resins after acetone washingviolet-visible（UV-Vis）spectrophotometer（V-650S,

further away from the fiber was not polymerized.

JASCO Corp., Tokyo, Japan）. Prior to the measure-

Table 2 shows the polymerization ratio of the resins

ment, each resin was stacked with glass plates andwith and without the fiber post in the two light irradia-

light-cured to 1 mm thickness. A cured resin plate wastion modes. The polymerization ratio of each resin wasused for these measurements.

significantly higher in mode-10 than in mode-F3 with

5

Statistical analysis

and without the fiber post. In the fiber-post groups with

Statistical differences in the polymerization ratio andmode-10, the polymerization ratio was over 96％ fordepth among the resins were confirmed using the Reach resin. Among these groups, there was no statisti-

software（Version R 3.6.3, The R Function, Vienna,cally significant difference between the polymerization

Austria）. Data were analyzed by performing a Student’s

ratios of each resin. However, in the fiber-post groups

t-test or one-way analysis of variance（ANOVA）, fol-

with mode-F3, the polymerization ratio of each resin

lowed by Tukey’s post-hoc test.

was relatively low: 90.5±3.6 for UN, 92.8±4.8 for BL,
85.2±3.7 for LC, 92.7±2.3 for FU, and 75.1±5.2 for MI.

Results

Table 3 shows the polymerization depth of the resins
with and without the fiber post in the two light irradia-

Figure 2 shows representative images of the polym-

tion modes. The polymerization depth showed the sameerized resin with and without the fiber post in the twotrend as the polymerization ratio. The polymerization

light irradiation modes. For the samples with no fiber

depth was greater in mode-10 than in mode-F3 for eachpost, in each irradiation mode, the apical area of eachresin. For the no-fiber-post groups with mode-F3, theresin was not polymerized. In particular, polymerization

polymerization depth significantly depended on theof the apical area was lower in the mode-F3 groups

resin type; the UN resin produced the highest polymer-

than in the mode-10 groups. In comparison, the fiber

ization depth, followed by the BL, FU, LC, and MI res-post improved resin polymerization in both the irradia-

ins. This result indicates that the polymerization depthtion modes. However, for the mode-F3 groups, the resin

depends on the resin type. The polymerization depth ofin the apical area was not partially polymerized, eventhe samples with fiber posts reached 15 mm regardlesswhen the fiber post was used. In particular, the resinof the light irradiation mode.

Dec, 2022

Polymerization of Light-curing Resins in a Root Canal Model

29

Polymerization ratio（％）of the resin with/without fiber post in two irradiation modes

Table 2Mode-F3
Resin

Mean

SD

Category

Without fiber post

UN
BL
LC
FU
MI

93.1
81.4
57.4
80.1
55.7

3.6
2.6
2.8
0.7
2.0

With fiber post

UN
BL
LC
FU
MI

90.5
92.8
85.2
92.7
75.1

3.6
4.8
3.7
2.3
5.2

＊

Mode-10＊Significant difference＊＊

Mean

SD

Category

A
B
C
B
C

98.3
97.6
96.7
98.3
74.7

1.5
1.3
1.0
0.6
1.8

F
F
F
F
G

Significant
Significant
Significant
Significant
Significant

D
D
D
D
E

96.6
97.8
97.7
97.0
96.2

3.6
0.6
0.5
1.6
1.2

H
H
H
H
H

Significant
Significant
Significant
Significant
Significant

n＝5, SD：standard deviation；＊Category：Same letters indicate that the values are not significantly different

as confirmed by Tukey’s test（p＜0.05）in each group. ＊＊Significant difference：statistical difference between
the polymerization ratio of each resin by mode-F3 and mode-10, analyzed using Student’s t-test（p＜0.05）.

Table 3Polymerization depth（mm）of the resin with/without fiber post in two irradiation modes
Mode-F3
Resin

Mean

SD

Category

Without fiber post

UN
BL
LC
FU
MI

15.5
14.1
10.2
12.9
8.9

0.3
0.5
0.5
0.2
0.3

With fiber post

UN
BL
LC
FU
MI

15.6
15.5
15.4
16.5
15.1

0.1
0.2
0.3
0.2
0.1

＊

Mode-10＊Significant difference＊＊

Mean

SD

Category

A
B
C
D
E

16.4
16.4
16.2
16.5
12.1

3.6
0.1
0.2
0.1
0.3

I
I
I
I
J

Significant
Significant
Significant
Significant
Significant

F
F
F
G
H

15.6
15.5
15.6
17.1
15.7

3.6
0.2
0.1
0.4
0.2

K
K
K
L
K

No significant
No significant
No significant
Significant
Significant

n＝5, SD：standard deviation；＊Category：Same letters indicate that the values are not significantly different

as confirmed by Tukey’s test（p＜0.05）in each group. ＊＊Significant difference：statistical difference between
the polymerization depths of each resin by mode-F3 and mode-10, analyzed using Student t-test（p＜0.05）.

100

Figure 3 shows the transmission spectra of eachat 465 nm, where the maximum intensity of the pen
light was located, followed by the UN, BL, MI, and FU
resins.

Discussion
This in vitro study investigated the effect of irradiation mode on the polymerization of different light-curing resins in a root canal model with and without afiber post. To maximize the effectiveness of the fiber
posts, i-TFC Luminous fibers, which have a high optical

Transmittance（％）

resin. The LC resin produced the highest transmittance

UN
BL
LC
FU
MI

90
80
70
60
50
40
30
20
10
0

200

Fig. 3400
600
Wavelength（nm）

800

Transmission spectra of each polymerization
resin

ODEP Vol. 2, No. 1

30transparency19）, were used as the post material. Our

observed under short-time light irradiation, mode-F3.

previous study revealed that the polymerization of theLight irradiation using a handheld light-curing unit

core resin with i-TFC Luminous fiber was significantly

was used in two modes29）: high intensity with a short

19）.

irradiation time and low intensity with a long irradia-

Therefore, i-TFC Luminous fibers could effectively

tion time. Therefore, this study examined light curing

facilitate resin polymerization in the apical root canal.

at mode-F3 and mode-10; the irradiation mode affected

higher than that with other types of fiber post

Conventionally, resin polymerization is estimated by

the polymerization of the resin. These irradiation

the degree of conversion, which can be obtained bymodes of the light-curing unit are similar to those of20‒22）orother commercial light-curing units29, 30）. In many funda-

spectroscopy. These methods help measuremental studies on the polymerization of resins using

the spectrum of the resin and derive the degree of con-light-curing units, experiments were performed underversion for the resin using the measured characteristic

similar irradiation intensity and time as in the present

peaks. This method quantitatively determines theirradiation modes29, 30）. Hence, the irradiation modes

polymerization degree of the resin. The drawback ofused in this study were considered acceptable.

means of Fourier transform infrared（FT-IR）
23‒25）

Raman

this method is that the degree of conversion is deter-

Every resin achieved a relatively higher polymeriza-

mined only for some spots on the sample, that is, ation ratio and depth in mode-10 than in mode-F3

small size with a thin layer on the sample surface. It isbecause the total excitation energy for mode-10 was

difficult to estimate the polymerization of the entire

much higher than that for mode-F3. The resins polym-

bulk resin core using FT-IR and Raman spectroscopy.

erized by light irradiation with mode-F3 showed insuffi-

To evaluate the polymerization of the bulk sample, thecient polymerization in the apical area of the root canal.

present study adopted the polymerization ratio andThis result implies that even high-intensity light irradi-

depth, which were obtained by washing the light-cured

ation cannot achieve sufficient polymerization underresin with acetone, followed by measuring its weightsuch short-duration irradiation. A similar result waschange and polymerization length. The present method

reported in a previous study in which short-time light

was demonstrated in a previous study on the polymer-

irradiation of less than 10 s did not fully polymerize theization of resins in a fiber-post/resin-core construction

resin, and so the partially polymerized resin did not. The method allows easy estimation of theexhibit optimal properties31）. Therefore, prolonged irra-

polymerized/unpolymerized locations in a bulky resin

diation is mandatory for complete polymerization of thecore, and hence is suitable for evaluating resin polymer-

resin in an apical root canal.

system19）

Resin polymerization depended on the transparency

ization in an apical root canal.
The present study used a root canal model with aof the resin; the highly translucent resin tended tocylindrical cavity 3 mm in diameter. This cavity size

exhibit a relatively higher polymerization ratio andwas chosen considering the maximum diameter of adepth. For instance, the UN resin had a relatively

human root canal; for example, the largest diameter ofhigher transparency, polymerization ratio, and depth

26）

the mandibular first premolar is reportedly 2.78 mm .

than the MI resin. Hence, resin transparency is one of

The present results clearly indicate that the fiber

the most important factors for ensuring polymerization

post facilitated the polymerization of the core resin in

in the root canal. In contrast, the FU resin exhibited a

the root canal in each light irradiation mode. In particu-

relatively higher polymerization ratio and depth,lar, the use of the fiber post helped improve the polym-

despite having a low optical transparency. This sug-

erization of the apical area. These results are in line

gests that there are other factors influencing thewith those of previous studies on the use of fiber posts

polymerization. Examples include the color, filler

27, 28）. Highly translucent fiber

amount, and chemical compositions of the initiator andposts can help better irradiate light into the apical root

resin monomer32, 33）. Further study on the fiber-post/

canal. As a result, free radical polymerization occurs via

resin-core construction system is required to clarify the

excitation of the photoinitiator. Based on our present

correlation between the polymerization of the resins

results, a remarkable improvement in the degree ofand their properties.

for resin polymerization

polymerization provided by the fiber post could be

Within the limitations of this in vitro study, weDec, 2022

Polymerization of Light-curing Resins in a Root Canal Model

revealed that a highly translucent fiber post and longirradiation mode can help improve the polymerizationof the apical area of light-curing resins. However, thepresent study was performed in vitro using an artificial
root canal model instead of human teeth, and the experimental conditions differed from clinical situations interms of humidity, the complex shape of the root canal,and salivary contamination. These environmental factors may adversely affect resin polymerization. In clinical practice, sufficient polymerization may not be
achieved even with long-time light irradiation using
highly translucent fibers. Both highly translucent fiber
posts and long-time light irradiation are essential factors for the long-term success of tooth reconstruction.
Furthermore, resin polymerization is related to its
mechanical and physicochemical properties. Fundamental research is required to reveal the correlation
between polymerization and the mechanical and physicochemical properties of resins.

Conclusions
Within the limitations of this study, we concluded
that the resin in an apical area cannot be fully polymerized by short-time irradiation, even with high intensity
and a highly translucent fiber post. Resin polymerization in a root canal requires both a long irradiation timeand a highly translucent fiber post.
Conflict of Interest
The authors have no conflicts of interest to declareregarding the publication of this paper.

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Dec, 2022

33

Original Article

ODEP 2（1）：33～39，2022

In vivo Assessment of Tissue Compatibility of Root Canal Sealer
Containing Surface Pre-reacted Glass-ionomer Fillers
Saori TANAKA, Hirofumi MIYAJI＊, Kayoko MAYUMI＊,
Yukimi KANEMOTO＊ and Erika NISHIDA＊
Division of General Dentistry Center for Dental Clinics, Hokkaido University Hospital＊

Department of Periodontology and Endodontology, Division of Oral Health Scicnce,
Hokkaido University Faculty of Dental Medicine

Abstract
Purpose: Surface pre-reacted glass-ionomer（S-PRG）fillers possess properties of releasing ions such as
fluoride, strontium, and borate ions, and exhibit bioactive effects. In this study, we assessed the in vivo preclinical effect of a sealer containing S-PRG filler（S-PRG sealer）. Specifically, we evaluated the inflammatory
response of periapical tissues to the use of S-PRG sealer in root filling, compared to that of sealers with othermain components（mineral trioxide aggregate（MTA）, resin, and noneugenol-zinc oxide（ZO））.
Materials and Methods: Under general and local anesthesia, premolar pulpectomies were performed in
10-month-old beagle dogs. After root canal preparation, cleaning, and drying, root filling was performed byapplying various sealers with gutta-percha master points. Periapical lucency was analyzed radiographically,
and the area of inflammatory cell infiltration with bone resorption was analyzed histologically. The inflammatory response of periapical tissues to S-PRG sealer was compared to those of clinically used sealers including
MTA（Pro Root MTA）, resin（AH Plus）, and ZO（Canals N）. Tissue specimens were prepared at 1 monthand 6 months after obturation.
Results: Periapical lucency was observed at 1 month in all groups. However, at 6 months, periapicallucency persisted with Canals N, but was infrequent with the other three fillers. In histological sections at 1
month, all groups showed similar levels of infiltration of inflammatory cells into the periodontal tissues adjacent to the root apical foramen. After 6 months, inflammation of the periodontal tissues was significantly
improved（p＜0.05）for all sealers except for Canals N.
Conclusion: The inflammatory response to S-PRG sealer at periapical tissues was milder than that to
Canals N. The S-PRG sealer may provide biocompatibility comparable with that of commonly and clinicallyused Pro Root MTA and AH Plus in the healing of the periapical region.
Key words: surface pre-reacted glass-ionomer（S-PRG）fillers, root canal sealer, biocompatibility, dog

Corresponding author: Saori TANAKA, Division of General Dentistry Center for Dental Clinics, Hokkaido University Hospital,
Kita 13, Nishi 7, Kita-ku, Sapporo 060‒8586, Japan
TEL: ＋81‒11‒706‒4266, FAX: ＋81‒11‒706‒4334, E-mail: tanaka@den.hokudai.ac.jp
Received for Publication: September 15, 2022/Accepted for Publication: October 24, 2022
DOI: 10.11471/odep.2022‒005

ODEP Vol. 2, No. 1

34

er-containing sealer25）. In the present study, we sought
to assess the clinical effects of S-PRG filler sealers in

Introduction

root filling. Specifically, we employed radiographic andEndodontic root canal sealers have been developed

histologic endpoints to evaluate the healing of periapicalfor root canal obturation in combination with a mastertissues when S-PRG sealer was used in root filling with

gutta-percha point. Many types of endodontic sealers,a master gutta-percha point, following the pulpectomy

variously composed of zinc oxide（ZO）, calcium hydrox-of dog premolars; comparisons were made with threeide, epoxy resin, and mineral trioxide aggregate（MTA）,

other types of sealers: Pro Root MTA, AH Plus, andhave been used clinically1‒5）. In addition to the sealing

Canals N.

ability and physical properties of the sealer, its biocompatibility is an important factor, given that endodontic

Materials and Methods

sealers directly contact periapical tissue at the apicalforamen after root canal filling6,7）.

1 ．Pulpectomy and root canal filling with root

Recently, a root canal sealer containing surface

canal sealers

pre-reacted glass-ionomer（S-PRG）filler（S-PRG sealer）

Four healthy female beagle dogs（age 10 months;

has been developed for endodontic use8,9）. The bioactiv-

weight, approximately 10 kg）were used as experimen-

ity of the S-PRG filler has previously been reported total animals（approved by the Animal Committee ofpromote tooth remineralization, while additionally pro-

Hokkaido University, Approval No. 13‒122）. The exper-

viding acid buffering capacity, antibacterial activities,

imental sites were mandibular second, third and fourthand caries prevention, via the releasing and recharging

premolar teeth, for a total of 48 root canals. For end-

abilities of six ions, including fluoride, sodium, stron-

odontic treatments, animals were administered general

10‒15）. In particular,

anesthesia with medetomidine hydrochloride（0.04 mg/

bioactive ions, including those of strontium, fluoride, and

kg Domitor, Nippon Zenyaku Kogyo, Koriyama, Japan）,

borate, have the potential to provide the root canalbutorphanol tartrate（0.15 mg/kg Vetorphale, Meiji

tium, aluminum, silicate, and borate

sealer with bioactive effects. In vitro experiments have

Seika, Tokyo, Japan）, and midazolam（0.15 mg/kg Dor-shown that strontium ions promote osteoblast prolifera-

micum injection 10 mg, Astellas Pharma, Tokyo, Japan）,

tion and inhibit bone resorption16‒18）. Fluoride has beenand local anesthesia with 2％ lidocaine hydrochloride

shown to stimulate osteoprogenitor cell growth inwith 1：80,000 epinephrine（Xylocaine Cartridge for

19）

vitro , and to induce the expression of genes involvedin bone formation and mineral density, reflecting an
20）

Dental Use, Dentsply Sirona, Tokyo, Japan）.
After preparation of the access cavity and removal of

improved bone remodeling activity in vivo . In addi-

pulp tissue, the root canal was spread with #40 K-files

tion, fluoride has been shown to exhibit antibacterial（Shofu, Kyoto, Japan）, while measuring with an apex

effects on gram-negative organisms21）, including bacte-locator（Root ZX, J Morita, Tokyo, Japan）until thericidal activity against Porphyromonas gingivalis inperiapical tissue was reached. Cleaning with 10％

22）. Furthermore, like fluoride, borate has beensodium hypochlorite（Neocleaner“Sekine,”Neo Dental

shown to exhibit antibacterial and osteogenic

Chemical Products, Tokyo, Japan）and 3％ ethylenedi-

effects23,24）. Clinically, borate exposure has been usedaminetetraacetic acid（Smear Clean, Nippon Shika

previously for the treatment of eye irritation and dry-Yakuhin, Shimonoseki, Japan）was followed by drying

ness of the mouth, nose, and throat.

using sterilized paper points. The root canal was thenvitro

Endodontic sealer incorporating S-PRG fillers has

filled with one of the four types of sealers by insertion

been shown to release these ions for at least 90 days8）.

of a gutta-percha master point（Shofu）, as shown in

Therefore, S-PRG filler-containing root canal sealer

Figure 1. The choice of sealer for a given tooth was（S-PRG sealer）is expected to facilitate the healing of

assigned randomly. The following sealers were used inperiapical tissue, including alveolar bone. In rat subcu-

this experiment: S-PRG sealer（Shofu; S-PRG group）, Pro

taneous implantation tests evaluating sealer cyto-com-

Root MTA（Dentsply Sirona; MTA group）, Canals N

patibility, S-PRG filler sealers demonstrated lower（GC Showayakuhin, Tokyo, Japan; Canals N group）, andinflammatory reactions than those seen with silica fill-

AH Plus（Dentsply Sirona; AH Plus group）（Table 1）.

Dec, 2022

In vivo Assessment of Tissue Compatibility of S-PRG Sealer

A

B

35

C

Fig. 1 Application of S-PRG sealer to root canal of premolar（A）S-PRG sealer after mixing. Scale bar：2 mm.
（B）The gutta-percha was coated with sealer. Scale bar：5 mm.

（C）Root canal filling with gutta-percha master point with adequate application of
S-PRG sealer. Scale bar＝5 mm.
S-PRG：surface pre-reacted glass-ionomer.
Table 1

Root canal sealers used in this study

Product

Composition

S-PRG sealer

Powder：zinc oxide-based inorganic compound filler, S-PRG filler, additive
Liquid：poly carboxylic acid derived, water, others

Pro Root MTA

Powder：calcium oxide, silicon dioxide, bismuth oxide, aluminum oxide, others
Liquid：purified water

Canals N

Powder：zinc oxide, rosin, bismuth subcarbonate, barium sulphate, yellow pigment
Liquid：fatty acid, propylene glycol

AH Plus

Paste A：epoxy resin, calcium tungstate, zirconium oxide, silica
Paste B：diphenyldiamine, aminoamadantane, tricyclodecanediamine, calcium tungstate,zirconium oxide, silica, silicon oil

S-PRG：surface pre-reacted glass-ionomer；MTA：mineral trioxide aggregate

The cavities were temporarily sealed with glass iono-

inflammatory response, the area of inflammatory cellmer cement（Base Cement, Shofu）. Dental X-ray images

infiltration with bone resorption around the root apexof the periapical tissues were obtained at 1 month and（inflammatory cell infiltration area）was measured using

6 months after obturation. In four groups, 6 roots each

image analysis software（Image J 1.14, National Insti-

at 1 month and 6 months postoperatively（48 roots intutes of Health, Bethesda, MD, USA）.

total）were used.
2 ．Histologic assessments

3 ．Statistical analysis
The means and standard deviations of inflammatory

After the imaging at 1 month and 6 months, the dogs

cell infiltration area were calculated for each of the four

were euthanized with an overdose of sodium pentobar-

groups. Differences among the groups were analyzed

bital（Somnopentyl, Kyoritsu, Tokyo, Japan）underusing two-tailed one-way ANOVA with Scheffé’s post

general anesthesia. After perfusion fixation in 10％ neu-

hoc test. Statistical analysis was performed using thetral phosphate-buffered formalin solution, tissue blocks

SPSS software package（version 11.0; IBM Corporation,

including each tooth were removed, demineralized inArmonk, NY, USA）
. p-values of ＜0.05 were considered

10％ formic acid, and paraffin-embedded. Serial sections

statistically significant.

were prepared parallel to the tooth axis, subjected tohematoxylin and eosin（HE）staining, and observed
under a light microscope. To evaluate the degree of

ODEP Vol. 2, No. 1

36
S-PRG

MTA

Canals N

AH plus3 ．Measurement of inflammator y cell infiltrationarea
To evaluate tissue response at periapical tissues, the

1 montharea of inflammatory cell infiltration was measured at 1
month and 6 months postoperatively. The measuring
area of a representative sample in each group is shownin Figure 3, and the results are shown in Figure 4.
Inflammatory cell infiltration areas in the S-PRG,
MTA, Canals N, and AH Plus groups were 1.59±1.33
mm2 , 1.39±0.89 mm2 , 1.84±0.87 mm2 and 1.16±0.97

6 months

mm2 at 1 month, and 1.17±0.75 mm2, 0.80±0.42 mm2,
2.49±0.31 mm2 and 1.08±0.47 mm2 at 6 months, respectively. No significant differences were observed among
the groups at 1 month. The inflammatory cell infiltration area in the S-PRG and MTA was improved at 6
Dental X-rays at 1 month and 6 months after
obturation with root canal sealers
Black arrows show apical lesion. Scale bar：1 mm.

Fig. 2

S-PRG：surface pre-reacted glass-ionomer；MTA：
mineral trioxide aggregate.

months compared to 1 month, but not significantly. Values in the AH Plus group were similar at 1 month and6 months. However, in the Canals N group, the inflammatory cell infiltration area was increased at 6 months
compared to that at 1 month. The 6-month values in the
Canals N group were significantly larger（p＜0.05）than
the 6-month values in the other three groups.

Results
Discussion
1 ．Radiographical obser vation
Representative radiographical images and periapicalIn this study, root canals of premolars in dog were

lucency frequency at 1 month and 6 months postopera-

filled using an S-PRG sealer after extraction of the pulp;

tively are shown in Figure 2 and Table 2, respectively.

S-PRG sealer was compared to three types of commer-

In all groups at 1 month, periapical lucency（X-ray

cial root canal sealers that are used clinically. At 1

transparency adjacent to the root apical foramen）was

month, apparent inflammatory cell infiltration in thefrequently observed. At 6 months, periapical lucency

periapical tissue was observed in all four groups. Therepersisted in the Canals N group, but was observed only

was no significant difference in inflammatory cell infil-

infrequently in the other three groups.

tration area among the four groups at this time point.

2 ．Histological obser vation

This observation may reflect irritation induced by the

Representative histological images at 1 month and 6

sealers where the sealers came in contact with periapi-

months postoperatively are shown in Figure 3. At 1

cal tissue, or the effects of root filing and root canalmonth in all groups, infiltration of inflammatory cells

spread. However, at 6 months, the Canals N groupthat were strongly stained with hematoxylin（i.e., lym-

exhibited a significantly larger inflammatory cell area

phocytes and mononuclear cells）was observed in thethan that seen in the other three groups. These results

periodontal tissues adjacent to the root apical foramen.

suggested that the S-PRG sealer, as well as Pro RootAt 6 months, the S-PRG, MTA, and AH Plus groups

MTA and AH Plus, ameliorate inflammation in periapi-

showed lower levels of inflammatory cell infiltration. In

cal tissues compared to Canals N. Similar results were

contrast, the Canals N group continued to show exten-

obtained by X-ray observation. Several previous reports

sive inflammatory cell infiltration at the later time

have shown the anti-inflammatory effects of strontium

point.

ions. S-PRG filler has been proven to release strontium,
which may be effective in suppressing inflammation8,9,16‒18,23,24）. Therefore, we postulate that, in the pres-Dec, 2022

In vivo Assessment of Tissue Compatibility of S-PRG Sealer
MTA

R

R

Canals N

AH plusRR

1 month

S-PRG

37

R

R

6 months

R

R

Table 2Frequency of periapical lucency（lucency cases/n＝6）

1-month
6-month

S-PRG

MTA

AH PlusCanals N

3
1

3
1

2
1

4
4

S-PRG：surface pre-reacted glass-ionomer；MTA：mineral trioxide aggregate

ent study, the S-PRG sealer released strontium ions at
the root apex, decreasing inflammation of the periapicaltissue. Previous work has shown that S-PRG fillers significantly promote cell viability of MC3T3-E1 cells（anosteoblast-like cell line）compared to silica filler25）, facilitating bone healing. On the other hand, it has beenreported that the solution eluted from S-PRG filler and
S-PRG sealer has antibacterial activity8,9,26‒29）. This prop-

Inflammatory cells infiltration area（mm2）Fig. 3 Histological findings in periapical tissues at 1 month and 6 months after obturation
Dashed lines indicate the extent of inflammatory cells infiltration. Scale bar：500 μm. Staining：hematoxylin-eosin
staining.
S-PRG：surface pre-reacted glass-ionomer；MTA：mineral trioxide aggregate；R：root.

4

1M

3

6M

＊

2
1
0

Fig. 4

S-PRG

MTA

Canals N

AH plus

Histological measurements of inflammatory cell
infiltration area（mm2）at 1 month and 6 months

after obturation with root canal sealers
n＝6 per group, mean＋standard deviation.
＊：p＜0.05 vs. other groups.
S-PRG：surface pre-reacted glass-ionomer；MTA：
mineral trioxide aggregate.

erty may maintain antibacterial effects in the root
canal. The results of the present study may reflect the

infiltration areas in the MTA group at 6 months werebiosafety and antibacterial properties of the S-PRG

less than those in the Canals N group at the same timesealer associated with the ion release effects of thispoint. We infer that MTA has superior biocompatibility

filler.

compared to Canals N. The other comparator, AH Plus,

MTA has been reported to have excellent hard tis-

is an epoxy resin-based sealer that has been reported tosue-forming abilities29） and persistent strong alkalinity,

have high sealing properties and to reduce coronal leak-as well as antibacterial properties and strong biocom-

age. However, a moderate inflammatory reaction has

30）

patibility . In the present study, the inflammatory cellbeen observed in implantation when AH Plus is usedODEP Vol. 2, No. 1

38

experimentally for direct filling in the jaw bone31）. In
the early stages of polymerization of AH Plus, a slightcytotoxicity（thought to result from elution of a small
amount of formaldehyde）has been reported, suggestingthat the irritation caused by formaldehyde in AH Plusis the source of this inflammation32）. However, while
many previous reports have shown the cytotoxicity of
AH Plus, the present study observed only slight cellular
infiltration at 1 month and 6 months.
Canals N is well-known as a biocompatible sealer, as
it does not contain eugenol, in contrast to Grossman’s
sealer. In the present study, teeth filled with Canals N
had significantly larger inflammatory cell infiltration

related to this article.
Ethics Approval
Animal experiments were conducted in accordance withthe regulations on animal experiments of Hokkaido University, with the approval of the Animal Committee of Hok. Experiments on
kaido University（Approval No. 13‒122）
animals were carried out in accordance with relevant guidelines and regulations.
Acknowledgments
We thank Shofu Inc. for providing the S-PRG sealer. This
study was supported by Hokkaido University, which provided facilities and resources.

areas（compared to the other three sealers）, even at 6
months after obturation. Previously, a histological studyof Canals N implanted subcutaneously in rat tissue
detected an accumulation of inflammatory cells aroundthe Canals N, even after 35 days

33）

. It has also beenreported that disintegrated Canals N in the body stimulates the expression of interleukin（IL）-1β34）, a cytokine
that contributes to bone resorption activity35）. Thus,
Canals N may have an adverse effect on apical healingeven at 6 months after use.

Conclusion
The inflammatory response to S-PRG sealer wascompared, both radiographically and histologically, withthose of Pro Root MTA, Canals N, and AH Plus after
the root obturation of premolars in beagle dogs. The
results showed that X-ray transparency adjacent to theroot apical foramen（periapical lucency）was observed
at 1 month in all groups. At 6 months, periapicallucency persisted with Canals N, but was observed only
infrequently in the other three groups. In addition,
when assessed at 6 months, teeth filled with the S-PRG
sealer, as well as those filled with Pro Root MTA andAH Plus, exhibited significantly decreased inflammatory cell infiltration and bone resorption compared tothose filled with Canals N. These results suggested that
the S-PRG sealer may provide good biocompatibility,
comparable to those of the commonly and clinicallyused Pro Root MTA and AH Plus, in healing of theperiapical region.
Conflict of Interest
The authors confirm that there is no conflict of interest

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Improved osteogenesis of boron incorporated calcium

ODEP Vol. 2, No. 1

40

Original Article

ODEP 2（1）：40～48，2022

Maillard Reaction Product of Rare Sugar Allulose Decreases
Bacteria-derived and Chemically-prepared Hydrogen Sulfide
Shigeru YAMAMOTO, Nao TANIGUCHI1, Jun TAKATORI, Junko HATAKEYAMA,Sami OMAGARI, Kazuhiko YAMADA, Takao HIROFUJI2 and Masahiro YONEDA
Section of General Dentistry, Department of General Dentistry, Fukuoka Dental College
1

Section of Oral Public Health, Department of Preventive and Public Health Dentistry, Fukuoka Dental College
2

Professor emeritus, Fukuoka Dental College

Abstract
Purpose: Oral malodor, which concerns many people, is associated with volatile sulfur compounds（VSCs）
produced by periodontopathic bacteria such as Porphyromonas gingivalis. Maillard products are used in thefood industry to decrease hydrogen sulfide（H2S）, which disturbs the flavor and taste of food. In the present
study, we planned to apply Maillard reaction products for reducing VSCs to meet the needs demand ofpatients who want to rapidly decrease oral malodor.
Methods: The effect of maple sugar solution（a Maillard reaction product）on VSC decrease was examinedby adding it to sonicated extract of P. gingivalis. Next, the rare sugar allulose was used for the Maillard
reaction（to generate Maillard-allulose）, because allulose has no calories and does not cause dental caries. The
effect of Maillard-allulose on VSC decrease was examined by adding it to P. gingivalis-derived and chemically-prepared H2S. After 1 min, headspace air was collected and the concentration of H2S was measured using
the Oral Chroma portable gas chromatography system. Separately, headspace air from a mixture of sodiummono-hydrogen sulfide and Maillard-allulose was analyzed by a solid phase micro extraction method with gas
chromatography-mass spectrometry.
Results: Both maple sugar and Maillard-allulose decreased bacteria-derived and chemically-prepared H2S.
New material seemed to be generated after mixing sodium mono-hydrogen sulfide and Maillard-allulose, indicating that H2S may have bound to a component of Maillard-allulose, resulting in loss of volatility of H2S.
Conclusion: Both maple sugar and Maillard-allulose had H2S-decreasing activity. Maillard-allulose is not
associated with dental caries and obesity, so it may be a good candidate for use in foods for decreasing oralmalodor.
Key words: oral malodor, Maillard reaction, rare sugar, allulose

Corresponding author: Masahiro YONEDA, Section of General Dentistry, Department of General Dentistry, Fukuoka Dental
College, 3‒1‒1 Tamura, Sawara-ku, Fukuoka 814‒0193, Japan
TEL: ＋81‒92‒801‒0411, FAX: ＋81‒92‒801‒4909, E-mail: yoneda@fdcnet.ac.jp
Received for Publication: September 15, 2022/Accepted for Publication: October 24, 2022
DOI: 10.11471/odep.2022‒006

Dec, 2022

Decrease of Hydrogen Sulfide by Maillard Reaction Product of Rare Sugar Allulose

41

biotic period compared with the placebo period13）.

Introduction

However, there is a weakness associated with probiotic treatment: it aims to improve the oral microbiota,

Many people worry about oral malodor1,2）, and theso it takes time to obtain good results and does not help

number of such patients seems to be increasing

patients who want to rapidly decrease oral malodor.

because of the wearing of masks to prevent infection3）.

Thus, we added green tea catechins to the probiotic

Oral malodor is associated with volatile sulfur com-tablet14）. The probiotic tablet containing green tea cate-

pounds（VSCs）produced by periodontopathic bacteria

chins was effective in controlling cariogenic and peri-such as Porphyromonas gingivalis and Tannerella for-odontopathic bacteria. It also lowered the concentration

sythia4）. These bacteria, which are included among theof methyl mercaptan, a VSC. However, although the5）

red complex bacteria , have high proteinase activity

VSC-decreasing effect was statistically significant, theand degrade proteins in the oral cavity, resulting inchange was not so drastic and patients may not notice

VSC production6）.

the effect. Therefore, we tried another strategy of

To decrease oral malodor, maintenance of oral healthby proper mouth cleaning is important. Mechanicaldecreasing the amount of hydrogen sulfide（H2S）, which
is another major VSC in oral malodor.

plaque control, such as by using a toothbrush, interden-

The Maillard reaction is an amino-carbonyl reaction.

tal brush, dental floss, and tongue scraper, is the first

It is a non-enzymatic browning reaction, which plays anline of prevention. Chemical plaque control, such as

essential role in food processing to improve the appear-

mouth rinse and dentifrice, used in addition to mechani-

ance and functional properties of food15）. The Maillard

cal oral hygiene procedures, is helpful in decreasing

reaction product is also known to decrease H2S pro-oral infectious diseases. Many studies have supported

duced in the food industry; H2S disturbs the taste andsignificant plaque decrease by use of chemical plaqueflavor of food15）. We considered using a Maillard reac-

control measures7）. However, the side effects and safetytion product, maple sugar, to decrease H2S in oral mal-of these measures are often of concern. For example,

odor. However, maple sugar may cause dental caries, so

chlorhexidine, the bactericidal agent that has been most

instead we considered using a rare sugar to control oral

studied and is recognized as the most effective formalodor.
“Rare sugar”is a comprehensive term forinhibiting plaque and preventing gingivitis, periodonti-

monosaccharides and sugar alcohols that are rare intis, and oral malodor, has several adverse effects,

nature. Strategies to produce rare sugars using epime-

including extrinsic tooth staining, calculus build-up,

rases have recently been reported by Kagawa Univer-transient taste disturbance, and effects on the oralsity16）. Rare sugars cannot be used by organisms formucosa8）.

their metabolism; indeed, they are known to inhibit car-Thus, we have been examining safer methods toiogenic bacteria17）. Allulose is a rare sugar, and as an

decrease oral malodor, one of which is the use of probi-

additional advantage, it is non-calorific, which is import-

otics. Probiotics have traditionally been used to treat

ant for people who are concerned about their visceraldiseases related to the gastrointestinal tract. However,fat or blood sugar level18）. Allulose can be chemically

recently, many studies have investigated the effects ofproduced by epimerization of D-fructose, and its priceprobiotic bacteria on oral health9）. Several clinical trials

has decreased in recent years19）. Therefore, we usedhave reported that regular consumption of Lactobacil-

allulose for the Maillard reaction to generate Mail-

lus salivarius WB21 decreases periodontitis and oral

lard-allulose, aiming to decrease levels of H2S.

malodor10‒13）. The use of oil drops containing L. salivar-

ius WB21 resulted in an improvement in periodontal

Materials and Methods

condition and a decrease in oral malodor in a randomized clinical trial12）. A 14-day, double-blind, placebo-con-

1 ．Bacterial strain and culture conditions

trolled, randomized crossover trial of tablets containing

P. gingivalis ATCC 33277 was maintained on CDC

L. salivarius WB21 in patients with oral malodor

anaerobic blood agar（Becton Dickinson, Cockeysville,

resulted in a significant decrease in the concentration of

MD, USA）in an anaerobic atmosphere（80％ N2, 10％

VSCs and the average probing pocket depth in the pro-

H2 , 10％ CO2）
, and inoculated into tryptic soy brothODEP Vol. 2, No. 1

42（Difco Laboratories, Detroit, MI, USA）supplemented
with hemin（5μg/ml, FUJIFILM Wako Pure ChemicalCo., Osaka, Japan）and menadione（1μg/ml, FUJIFILM

7 ．Heat-concentration and dialysis of Maillardallulose
Maillard-allulose was concentrated by heating untilthe volume became half of the original. The concen-

Wako Pure Chemical Co.）.
2 ．Preparation of sonicated extracts of P. gingi-

trated Maillard-allulose solution was then dialyzed
against distilled water using a Spectra/Por membrane

valis cells
Sonicated extracts（SE）of P. gingivalis cells were（molecular weight cutoff 6‒8,000; The Spectrum Com-

prepared as reported previously20）. Briefly, P. gingivalis

panies, Charlotte, NC, USA）, and reconcentrated to thecells in the late logarithmic growth stage in tryptic soy

original volume by heating.

broth were harvested by centrifugation and washedwith phosphate-buffered saline（PBS）. Five hundred

8 ．Experiment to assess the possible mechanismof H2S reduction by Maillard-allulose

micrograms of bacterial cells were suspended in 5 ml ofHeadspace air from solution containing sodium

PBS, and the cells were disrupted by sonication on ice.

mono-hydrogen sulfide, Maillard-allulose, or a mixture of

Intact cells were removed by centrifugation.

sodium mono-hydrogen sulfide and Maillard-allulose

3 ．Measurement of P. gingivalis-derived VSCs

was collected and adsorbed to Stableflex Fiber Carb/

One milliliter of distilled water or maple sugar was

PDMS 85μm（Merck KGaA, Darmstadt, Germany）byadded to 1 ml of P. gingivalis SE in a plastic tube witha solid phase micro extraction（SPME）method23）. Thea soft cap, and vortexed at room temperature. After 1

air component of each sample was analyzed by gas

min, the cap of the tube was penetrated with a needle

chromatography-mass spectrometry（7890A GC system,

attached to a plastic syringe, and the headspace air was

Agilent Technologies Japan, Ltd., Tokyo, Japan）con-collected. The level of H2S was measured using a porta-

nected to a VF-WAXms（Agilent CP9205）column

ble gas chromatography system, Oral Chroma CHM-2（Nissha FIS, Osaka, Japan） .

21）（Agilent Technologies Japan, Ltd.）.
9 ．Statistical analysis

4 ．Maillard reaction of allulose

To analyze the decrease of H2S by maple sugar, Stu-

Allulose（final conc. 0.1 M; FitLane Nutrition, Conroe,

dent’s t-test was applied. To compare the decrease of

TX, USA）was mixed with various amino acids（final

H2S by various materials and test the homogeneity ofconc. 0.1 M; FUJIFILM Wako Pure Chemical Co.）andvariance, the Levene test was applied. Analysis of vari-

the pH was adjusted to 9.0 with sodium hydroxide if theance was also performed. For multiple comparison,

pH of the solution was ＜9.0. The solution was heated in

Tukey’s test was applied.

a water bath at 95℃ for 90 min22）. Completion of theMaillard reaction was confirmed by the browning of theResults

allulose solution. The reaction product is referred to
1 ．Decrease of P. gingivalis-derived VSCs by

hereafter as Maillard-allulose.
5 ．Decrease of P. gingivalis-derived H 2 S bymaple sugar

maple sugar suspension and Maillard-allulose

Three milliliters each of distilled water and either

Maple sugar solution（0.3 g/ml; Maple-Farms Japan

maple sugar suspension（final conc. 0.3 g/ml）or glucose

Co., Osaka, Japan）or Maillard-allulose solution was

suspension（final conc. 0.3 g/ml）were added to P. gin-

added to P. gingivalis SE suspension. After 1 min, head-givalis SE suspension. After 1 min, the headspace air

space air was collected with a needle and the concen-

was collected and the H2S concentration was measured

tration of H2S was measured using the Oral Chromausing the Oral Chroma system（Figure 1）. Maple sugar

CHM-2 apparatus.

suspension significantly decreased the H2S concentra-

6 ．Treatment of chemically-prepared H 2 S bytion in the P. gingivalis SE suspension tube（p＜0.05）.
Maple sugar decreased the concentration of H2S fromMaillard-allulose
Chemically-prepared H2S was generated by dissolv-

ing NaHS・nH2O（FUJIFILM Wako Pure Chemical Co.）－5

in distilled water. It was diluted to 10 ％.

just after addition of the maple sugar suspension. The
H2S concentration then increased slightly over 40 min.
The amount of H2S in the control started to decline
after 30 min（Figure 2）.

Decrease of Hydrogen Sulfide by Maillard Reaction Product of Rare Sugar Allulose

5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0

43

m

ap
le

co

su

nt
ro

gl
uc
o

ga
r

se

＊

l

H2S（ng/ml）

Dec, 2022

Fig. 1 Effect of maple sugar solution on H2S concentration
An equal amount of maple sugar solution or glucose
solution（0.3 g/ml）was added to Porphyromonas gingivalis sonicated extract suspension. After 1 min, headspace air was collected and the H2S concentration wasmeasured using the Oral Chroma system. Maple sugar
suspension significantly decreased the H2S concentra-tion（p＜0.05）. Data show the means and standard
deviations for three samples.

H2S（ng/ml）

30
25Fig. 3 Color change after Maillard reaction
Allulose（final conc. 0.1 M）was mixed with L-arginine（final conc. 0.1 M）and the pH was adjusted to 9.0 withsodium hydroxide. The solution was heated in a water
bath at 95℃ for 90 min. The mixture of allulose andL-arginine was initially colorless（right）, but after heating, it became brown（left）, which indicates the completion of the Maillard reaction. The results shown are
representative data for six similar sets of experimental
data.

orless. It became brown after heating at 95℃ for 90

20

min, and the Maillard reaction was considered to be

15completed.
3 ．Combination with various amino acids

10

Various amino acids were mixed with allulose for the

50

Maillard reaction. Among the 11 tested amino acids, the
0

10

20

30

40（min）

Fig. 2 Time course of H2S concentration
An equal amount of maple sugar solution（0.3 g/ml）or distilled water was added to P. gingivalis
sonicated extract suspension. At each time point,
headspace air was collected and the H2S concentra-tion was measured using the Oral Chroma system.
The H2S concentration decreased just after addi-

tion of maple sugar suspension. The control H2S

concentration（blue line）began to decrease after
30 min（no statistical analysis）. The H2S concentra-tion with maple sugar solution（orange line）waslow but increased slightly over 40 min. The results
shown are representative data for four similar setsof experimental data.

Maillard reaction product obtained using arginine had
the greatest effect in decreasing the concentration of
H2S, and the amount of H2S became 0 in this experimental condition. L-serine, L-alanine, and L-cysteine had
no H2S-decreasing effect（Figure 4）.
4 ．Confirmation of necessity of Maillard reactionfor H2S reduction
Maillard-allulose produced with L-arginine showed astrong decreasing effect on H2S, but an unheated mixture of allulose and L-arginine had no effect（p＜0.01）,
indicating that the heating process（i.e., the Maillard
reaction）is necessary for H2S decrease（Figure 5）.
5 ．Ef fect of heat-concentration and dialysis on
the effect of Maillard-allulose
Maillard-allulose was concentrated by heating. The
concentrated Maillard-allulose showed a strong decreas-

2 ．Maillard reaction of allulose

ing effect on the level of H 2 S, indicating that the

An illustration of Maillard-allulose is shown in Figure

reagent was heat-stable. Even after dialysis against dis-

3. A mixture of allulose and L-arginine was almost col-tilled water, it retained a strong effect（Figure 6）.

ODEP Vol. 2, No. 1

120

60

100

50

H2S（ng/ml）

80
60
40
20

40
30
20
10

Allulose（final conc. 0.1 M）was mixed with various
amino acids（final conc. 0.1 M）and the pH was adjusted
to 9.0 with sodium hydroxide if the pH of the solution
was ＜9.0. The resulting solution was heated in awater bath at 95℃ for 90 min. Among the 11 aminoacids tested, the product with L-arginine showed thestrongest reducing effect on H2S（no statistical analy-

sis）. The results shown are representative data forthree similar sets of experimental data.

Fig. 6

d
ly
ze
di
a

e
d－
all
ul
os
e
co
nc
en
tr
at
ed

os
su
cr

Effect of heat-concentration and dialysis of
Maillard-allulose on decrease of H2S

Maillard-allulose was concentrated by heating untilthe volume became half of the original. The concentrated Maillard-allulose was then dialyzed against distilled water, and reconcentrated to the original volume
by heating. Heated Maillard-allulose decreased theconcentration of H2S, indicating that the effect was

heat-stable. Dialysis had no effect on the activity, indicating that large molecules in the Maillard-allulose
reaction mixture are responsible for H2S reduction（nostatistical analysis）. The results shown are representative data for four similar sets of experimental data.

＊

80
70
60
50
40
30
20
10
0

terns of H2S alone and Maillard-allulose were different.
Then, H2S and Maillard-allulose were mixed and theheadspace air was subjected to gas chromatography.
The peak pattern of this mixture mostly looked like a

gin
ar
L－
se
＋
ulo
all

M

ai
lla

co

rd
－a
l

lu

nt

lo

ro

ine

se

mixture of the two individual peak patterns. However, al

H2S（ng/ml）

＊

gl
uc
o

ail
lar

Effect of various amino acids on reduction of
H2S

M

as

Fig. 4

se

0

pa
r

d
tic w
L－ ac
pr id
o
L － linese
L－ r
ar ineL－ gi
iso ninle eu
L－ cinal eaL－ nineva
L－ lingl eyL－ cineL－ lyscy ineL－ stehi ine
st
id
in
e

0

dw

H2S（ng/ml）

44

Fig. 5 Effect of Maillard reaction on reduction of H2SAllulose（final conc. 0.1 M）was mixed with L-arginine（final conc. 0.1 M）and the pH was adjusted to 9.0
with sodium hydroxide. The solution was heated in awater bath at 95℃ for 90 min. Control solution was notheated. Maillard-allulose significantly decreased theH2S concentration, but the unheated mixture of allu-

lose and L-arginine had no effect, indicating that Maillard-allulose is necessary for the H2S reduction（p＜new big peak, which was above the scale limit, wasgenerated at a retention time of around 21.6 min. An
analysis of the retention time and a database search by
Kumamoto Industrial Research Institute indicated that
there was a 97％ probability that the new peak wascaffeine.

Discussion
The Maillard reaction is necessary in the food industry because it can decrease H2S, which worsens the flavor of various foods. This fact prompted us to think of0.01）. The data show the means and standard deviations for eight samples.

using a Maillard reaction product to decrease oral mal-

6 ．SPME-gas chromatography analysis

expected. The effect was observed just after addition to

The results of SPME-gas chromatography-mass spec-

sonicated bacterial extract. However, continuously tak-trometry analysis are shown in Figure 7. The peak pat-ing maple sugar to decrease oral malodor could lead toodor. Maple sugar, which is a Maillard reaction product,
showed a strong decreasing effect on H2S, as we hadDec, 2022

Decrease of Hydrogen Sulfide by Maillard Reaction Product of Rare Sugar Allulose

45

A

B

new peakC

Solid phase micro extraction-gas chromatography-mass spectrometry
analysis
Headspace air from solution containing sodium mono-hydrogen sulfide, Maillard-allulose, or a mixture of sodium mono-hydrogen sulfide and Maillard-allulose was collected and adsorbed to Stableflex Fiber Carb/PDMS 85 μm by a solid phase micro
extraction method. The air component of each sample was analyzed by gas chromatography-mass spectrometry using a VF-WAXms column. A：Peak pattern of headspace air of solution of sodium mono-hydrogen sulfide alone. B：Peak pattern ofheadspace air of solution of Maillard-allulose alone. C：Peak pattern of headspace airof mixture of sodium mono-hydrogen sulfide and Maillard-allulose. The arrow indicates the new peak（above scale limit）that was generated on mixing sodiummono-hydrogen sulfide and Maillard-allulose.
Fig. 7

dental caries and obesity, so it is not appropriate to use

because simply mixing allulose and L-arginine without

maple sugar for oral malodor control.

heating did not decrease H2S. The H2S concentration of

Next, we considered using a rare sugar instead of

the P. gingivalis SE-maple sugar mixture was low, butmaple sugar to control oral malodor. Rare sugars are

it slightly increased over 40 min. The reason is not18）

non-calorific17）and do not cause dental caries . There-

fore, we used allulose for the Maillard reaction, to generate Maillard-allulose to decrease levels of H2S.

clear yet, but the binding between H2S and maple sugar
may have been reversible; further study is required.
To clarify the mechanism of H2S reduction, we per-

The Maillard reaction is completed by mixing sugars

formed SPME-gas chromatography-mass spectrometry

and amino acids under alkaline conditions, followed byexperiments23）. Headspace air from samples containing

heating. The proportion of sugar and amino acid affects

H2S alone and Maillard-allulose alone showed different

the viscosity of the reaction product, so we used condi-

peak patterns. The peak pattern of the mixture of H2Stions that did not become too sticky. Various kinds ofand Maillard-allulose mostly looked like a mixture ofamino acids may be used for the Maillard reaction, so

those two peak patterns, but an intense new big peak

we first determined the most suitable combination for

was generated when H2S and Maillard-allulose weredecreasing H2 S. We tested inexpensive amino acids

mixed. Analysis of the retention time and a databasewith a view to applying our approach clinically in the

search by Kumamoto Industrial Research Institute indi-

future. Among the 11 amino acids used in this experi-cated that there was a 97％ probability that the newment, the Maillard reaction product of L-arginine waspeak was caffeine. These results indicate that somefound to be the most effective in decreasing the level ofmolecule in Maillard-allulose combined with H2S to form

H2S, so we used L-arginine for further experiments.

a new product. By combining with this molecule, H2S is

The Maillard reaction was shown to be necessary,considered to have lost its volatility. To confirm thisODEP Vol. 2, No. 1

46

hypothesis, identification of the putative caffeine-like

effective in decreasing the concentration of H2S. The

reaction product is under way.

Maillard reaction was required to achieve this effectThis work has some limitations. Even in the controls,

because an unheated mixture of allulose and L-arginine

the level of H2S began to decrease during the assay,

did not decrease H2 S. New material may have been

due to evaporation. The Oral Chroma measurement

generated after mixing sodium mono-hydrogen sulfide

took ＞8 min for each assay. Thus, we could not repeat

and Maillard-allulose, indicating that some molecule inassays many times and could not present standard

Maillard-allulose combined with H 2 S to form a new

deviations in some experiments. To overcome thisproduct, which reduced the volatility of H 2 S. Fromproblem, we repeated each experiment under the samethese results, Maillard-allulose may be a good candidate

conditions and confirmed reproducibility. We need tofor use in foods for decreasing oral malodor.

find new assay methods that allow quicker measurement.

Conflict of Interest

Oral malodor sometimes affects patients’ quality oflife and disturbs communication with others

24,25）. Some

The authors declare that there is no conflict of interest in
this study.

patients use antibiotics and disinfectants to decrease
oral malodor26）, and this can be effective for a time.
Periodontitis is caused by infection with periodontal

Ethical Statement
Not applicable.

pathogens, but periodontal tissue damage is thought tobe caused by various factors27）. Continuous use of disin-

Acknowledgments

fectants may give rise to side effects such as bacterial

We express our gratitude to Dr. Shigeru Hayakawa（professor emeritus of Kagawa University）for his valuableadvice about the experiments. We thank Mr. Tadashi
Masuda and Mr. Kentaro Masuda of Zen Corporation for
their help and discussions. We also thank Dr. Nami Mimaki
and Dr. Takao Sato of Kumamoto Industrial Research Institute for technical assistance. This work was in part supported by Grants-in-Aid for Scientific Research（No.
20K09886 and 18K09781）from the Ministry of Education,
Culture, Sports, Science and Technology（MEXT）. We thankEdanz（https://jp.edanz.com/ac）for editing a draft of thismanuscript.

resistance and microbial substitution28）. Thus, we arefocusing on non-disinfectant control of oral malodor. In
our previous experiments, the use of probiotics wasfound to improve the oral microbiota and decrease oralmalodor, because oral malodor is strongly associatedwith the oral microbiota29,30）. Gut microbiota are also
related to oral conditions, including oral malodor31,32）.
However, improvement of the oral microbiota by using
probiotics is slow. Zinc ions are also known to effectively decrease oral malodor33）, but care is required to
avoid excessive intake.
Maillard-allulose has a good taste and little effect on
obesity34）or caries formation. If the new method developed here using Maillard-allulose to decrease levels of
H2S is successful in clinical application, oral malodor
could be decreased instantly. We are also considering
combining Maillard-allulose and probiotics, which would
contribute to both improvement of the oral microbiota
and rapid decrease of oral malodor.

Conclusions
Maple sugar solution decreased P. gingivalis-derived
H2 S. The Maillard reaction product with rare sugar
allulose decreased P. gingivalis-derived H2S and chemically-prepared H2S. Among various amino acids, theMaillard reaction product with L-arginine was most

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Dec, 2022

49

Original Article

ODEP 2（1）：49～59，2022

Study on Sealability of Dentinal Tubules Irradiated by Nd: YAG Laserwith TiO2 Using a Model of Hypersensitive Dentin
Tomohiro NAKATA, Kenzo YASUO, Yuto MORIKAWA,
Naohiro IWATA, Hiroaki TANIMOTO, Kazushi YOSHIKAWA
and Kazuyo YAMAMOTO
Department of Operative Dentistry, Osaka Dental University

Abstract
Purpose: In recent years, there have been reports on the application of Nd: YAG laser（the laser）combined, a photoreactive agent, to dental treatment, but there have been no reports onwith titanium dioxide（TiO2）
its use for dentin hypersensitivity for the purpose of high reactive level laser therapy. In this study, to investigate the transmittance of the laser combined with TiO2 through dentin, the transmitted energy of the laser
was measured. Furthermore, to investigate the sealing ability of the laser on dentinal tubules, Nd: YAG laser
combined with TiO2 was irradiated to a model dentin affected with hypersensitivity, and the dentin permeability inhibition ratio（permeability inhibition ratio）was measured.
Methods: STREAK-1（ALTECH）was used as the laser system. Human extracted teeth were used as test
teeth, and dentin disc specimens of 1.0 mm in thickness were prepared.
Experiment 1: Examination of laser penetration through dentin
The irradiation conditions were 50μs-50 mJ-10 pps, 100μs-100 mJ-5 pps, and 200μs-300 mJ-5 pps, and theirradiation time 5 seconds. The laser was irradiated through one to five dentin discs, and the transmittancewas calculated. In the same way, the laser was irradiated together with TiO2 solution, and the transmittancewas calculated for a single dentin disc.
Experiment 2: Examination of dentinal tubule sealing ability of laser combined with TiO2 solution
The irradiation conditions were 50μs-50 mJ-50 pps, 100μs-100 mJ-20 pps, 100μs-100 mJ-99 pps, and 200
μs-300 mJ-20 pps, and the irradiation time was set to 10 s, 20 s, and 30 s. The specimens were connected tothe measuring device and laser was irradiated with TiO2 solution. The permeability inhibition ratio was measured, and the dentin disc surface was observed by SEM.
Results: Laser transmittance into dentin significantly decreased with increasing dentin thickness under all
the conditions. In addition, the transmittance of the laser significantly decreased under all the conditions
when the TiO2 solution was combined.
Regarding the sealing ability of the laser combined with TiO2 solution on dentinal tubules, the permeability
inhibition ratio increased significantly with increasing irradiation time at 50μs-50 mJ-50 pps and 100μs-100
mJ-20 pps. No significant difference was observed in the permeability inhibition ratio at 100μs-100 mJ-99 pps
and 200μs-300 mJ-20 pps. SEM observation of the surface of the specimens showed that the dentinal tubules
were sealed by cohesive structures under all the conditions.
Conclusion: The investigation indicates that Irradiation with Nd: YAG laser combined with TiO2, a photoreactive agent, attenuates the penetration of the laser into dentin and it is effective for sealing dentinal
tubules.
Key words: Nd: YAG laser, TiO2, dentin hypersensitivity
Corresponding author: Kenzo YASUO, Department of Operative Dentistry, Osaka Dental University, 8‒1, Kuzuha-hanazono-cho, Hirakata, Osaka 573‒1121, Japan
TEL: ＋81‒72‒864‒3077, FAX: ＋81‒72‒864‒3177, E-mail: yasuo-k@cc.osaka-dent.ac.jp
Received for Publication: September 15, 2022/Accepted for Publication: October 26, 2022
DOI: 10.11471/odep.2022‒007

ODEP Vol. 2, No. 1

50

TiO2 was performed on a hypersensitivity-affected dentin model, and the dentin permeability inhibition ratio

Introduction（permeability inhibition ratio）was measured.

In various branches of dentistry, many fundamental
studies and clinical reports on various types of lasers

Materials and Methods

have been conducted, and lasers are now used in various treatments such as conservative restorative proce-

STREAK-1（400μm diameter quartz fiber, ALTECH

dures, endodontic procedures, periodontal procedures,Co., Ltd., Yamanashi, Japan, Fig. 1）was used as the Nd:and surgical procedures1‒7）. It has been reported thatYAG laser（the laser）unit. 10％ TiO2 solution was pre-Nd: YAG laser is effective for removing enamel andpared by adjusting 40％ TiO2 solution（Avant toothdentin8‒10）11）, imparting acid resistance to dentin , and11,12）

sterilizing gingiva and periodontal pockets

by using

titanium dioxide（TiO2）, a type of semiconductor, as aphotoreactive agent during irradiation.
Recent years have seen an increase in the number ofsurviving permanent teeth per person in Japan owing
to dental care based on minimal intervention and the13）

liquid XBS, Miyako Chemical Co., Ltd., Kyoto, Japan）
with distilled water. The density of TiO2 solution was
set based on the preliminary experiment and themethod of Waga, etc.25,26） TiO2 solution was dripped at
the rate of about 0.3 ml/s at the time of laser irradiation.
1 ．Dentin disc specimens

promotion of oral health care . At the same time, how-After extraction for therapeutic reasons at theever, increasing numbers of patients are suffering from

Department of Oral and Maxillofacial Surgery of ourdentin hypersensitivity, which is characterized by tran-

university hospital, caries-free healthy human molars

sient pain due to cold or rubbing pain14）.

frozen at －40℃ in saline solution were thawed imme-

The primary theory behind the onset of pain due todiately before the experiment and used as test teeth.

dentin hypersensitivity is the hydrodynamic theory,

Flat dentin surfaces were prepared by planing fromwhich posits that pain is caused by stimulation of free

the occlusal surface with a model trimmer. The root

nerve endings in the pulp by the movement of fluid in

was then cut near the cervical area and the pulp was

the dentinal tubules caused by external stimuli trans-removed. We polished it up to #600 with waterproof

mitted through the dentinal tubules opening on the sur-abrasive paper, and prepared a cylinder-shaped dentin

face of the dentin15）. Treatments for dentin hypersensi-

disc with an exposed dentin surface of 8.0 mm in diam-tivity include conservative therapies such as application

eter and 1.0 mm in thickness. A 10％ phosphoric acidof medicament, iontophoresis, laser treatment, and cov-

solution（Kishida Chemical Co., Ltd., Osaka, Japan）was

errage with adhesive materials. However, the cure rateapplied to the occlusal side of the dentin disc for 30 sec-of conservative therapy is reported to be about 60‒

onds, and the disc was rinsed under running water for

16）

70％ , and symptoms may recur. Laser treatment is a

5 seconds. Then, a 5％ sodium hypochlorite solution

less invasive method that involves the use of photo-（Kishida Chemical Co., Ltd.）
was applied to the pulp sidechemical and photothermal effects in biological interac-for 10 seconds, followed by a 5-second rinse under run-tions

17,18）. Pain relief and accelerated healing can bening water and a 5-minute ultrasonic rinse with distilledexpected with low reactive level laser therapy（LLLT）

water to open the dentinal tubules for use as dentin

for alleviating hypersensitivity and high reactive level

disc specimens.

laser therapy（HLLT）for sealing dentinal tubules byThis study was approved by the Ethics Committee ofprotein denaturation and coagulation using a surface-ab-

Osaka Dental University（Approval No.: Osaka Dental

sorbing laser19‒24）.

University Ethics Committee No. 110767）.

In this study, the transmitted energy of Nd: YAG
laser was measured to investigate the effects of dentin

2 ．Experiment 1: Review of laser penetration
through dentin

thickness and TiO2, a photoreactive agent, on the trans-

Table 1 shows the laser irradiation conditions. The

mittance of Nd: YAG laser through dentin. Moreover,irradiation conditions were set based on the prelimi-

with the aim of sealing the dentinal tubules on the den-nary experiment. A laser power meter（FieldMax Ⅱ,

tin surface, Nd: YAG laser irradiation combined with

Coherent Inc., California, USA）
was used to measure theDec, 2022

Sealability of Dentinal Tubules Irradiated by Nd: YAG Laser with TiO2

51

irradiation conditions were set based on the preliminary experiment.
As the experimental system, a model dentin affectedwith hypersensitivity was prepared based on themethod of Zennyu, etc.27）（Fig. 2）. A rubber ring with an
inner diameter of 6 mm was placed on the pulp side ofthe dentin disc specimen and sandwiched between theupper and lower stainless-steel holders to be used as
the specimen stage. The center of the specimen stage
was opened into a square with a diameter of 5 mm
（approximately 0.25 cm2 in area）to expose the surface
to be irradiated by the laser. Dentinal tubular fluid（DF）was prepared by diluting parent bovine serum（Invitrogen）fourfold with distilled water to reproduce
Fig. 1

Nd: YAG laser unit used in this study

the protein content of DF in a clinical setting28‒30）. After
filling the specimen stage with DF to prevent air from

irradiation energy.

entering, the DF was connected to a device that can

1 ）Effect of dentin thickness

specify the internal pressure, made according to theLaser was irradiated from a distance of 1.0 cm frommethod reported by Pashley et al.31）. The internal pres-the measured surface, and the energy was measured.

sure of the specimen stage was adjusted to 25 mmHg,

Measurements without the dentin disc were used aswhich is the same as the internal pressure in human

the control. Next, dentin specimens of 1.0 to 5.0 mm

dental pulp, to reproduce pulp pressure in a clinical set-

were prepared and placed on the measured surface

ting32‒35）. DF from the direction of the syringe was shutusing dentin discs, and the laser energy after transmis-

off with the three-way cock B, and the glass capillarysion through dentin specimens of different thicknesses

tube connected to the specimen stage and the experi-

was measured. Based on the dentin thickness, the spec-

mental device was connected. In this state, air was

imens were classified into groups DT0, DT1, DT2, DT3,

blown onto the exposed enamel surface of the central

DT4, and DT5, respectively. From the obtained values,

opening of the specimen stage for 60 seconds andthe transmittance of the laser was calculated according

allowed to stand for another 30 seconds, after which theto the thickness of the dentin specimen.

DF movement in the glass capillary tube was mea-

2 ）Effects of TiO2 solution

sured36‒38）. The air pressure was set to 0.3 MPa. Next,

The thickness of the dentin specimen was set to 1.0

the three-way cock B was adjusted to shut off the DF

mm. Dentin specimens were placed on the measured

flow to the glass capillary tube, and DF was adjusted so

surface and laser was irradiated from a distance of 1.0

that DF could flow only from the syringe to the speci-

cm from the surface. Then, laser energy transmitted

men stage. Next, the three-way cock A was adjusted tothrough the dentin specimens with and without TiO2

inject DF from the syringe to the pressure gauge andsolution was measured. The group without solution was

specimen stage, and the internal pressure was adjusted

designated as the E（－）group, and the group withto 25 mmHg. Laser was irradiated together with TiO2

solution was designated as the E（＋）group. The value

solution. As before laser irradiation, the specimen was

without solution was designated as the control, and the

air-blown for 60 seconds and allowed to stand fortransmittance of the laser was calculated.

another 30 seconds, then the amount of DF movement

Five measurement points were used. The calculated

was re-measured. The respective permeability inhibi-transmittance was statistically processed by a one-waytion ratios were measured from the amount of DF

analysis of variance and Tukey’s test（p＜0.05）.

movement before and after laser irradiation. Using the

3 ．Experiment 2: Review of dentinal tubule sealing abilities
Table 2 shows the laser irradiation conditions. The

amount of DF movement before treatment（x）and theamount of DF movement after treatment（y）, the ratios
were calculated as follows:

ODEP Vol. 2, No. 1

52
Table 1

Irradiation output

code
Condition 1
Condition 2
Condition 3

Iradiation time（s）

Pulse duration（μs） Pulse energy（mJ） Pulse rate（pps）

50‒10
100‒5
300‒5

50
100
200

Table 2code
Condition 1
Condition 2
Condition 3
Condition 4

Laser irradiation conditions in Experiment 1

50
100
300

10
5
5

5
5
5

Laser irradiation conditions in Experiment 2
Irradiation output

Fluence（J/cm2）

Pulse duration（μs） Pulse energy（mJ） Pulse rate（pps）

Iradiation time（s）
10
20
30

50‒50
100‒20100‒99300‒20

50
100
100
200

50
100
100
300

50
20
99
20

100
80
396
240

200
160
792
480

300
240
1,188
720

stainless-steel holder（upper）
specimen stage

dentin disc specimen
rubber ring

glass capillary tube

stainless-steel holder（lower）

DF：bovine serum

three-way cock B

glass injection syringe

three-way cock A

pressure gauge

Schematic diagram of a dentin hypersensitivity model
A rubber ring with an inner diameter of 6 mm was placed on the pulp side of the dentin
Fig. 2

disc specimen, inserted between the upper and lower stainless-steel holders, and used as a
specimen stage. The pressure in the specimen stage was adjusted to 25 mmHg, equal tohuman dental pulp, to reproduce the pressure in clinical practice.

Permeability inhibition ratio（％）
＝
｛
（x－y）
/x｝
×100
…（1）

a scanning electron microscope（JSM-5610LV, JEOL
Ltd., Tokyo, Japan）.

The number of specimens was 5. The calculated permeability inhibition ratios were statistically processed

Results

by one-way analysis of variance and Tukey’s test（p＜0.05）for the difference in irradiation time at each irradiation power and for the difference in irradiation
power at each irradiation time.
Each specimen was deposited with gold in accordance with the conventional method and observed with

1 ．Experiment 1: Review of laser penetration
through dentin
Figure 3 shows the results of laser transmittance fordifferent dentin thicknesses.
The inhibition ratio under irradiation condition 50‒10

100

100

a
50-10

80
60
40

b

20
0

0

c

d

d

e

1
2
3
4
5
Thickness of dentin（mm）

Transmittance of laser（％）

Transmittance of laser（％）

Sealability of Dentinal Tubules Irradiated by Nd: YAG Laser with TiO2

Transmittance of laser（％）

Dec, 2022

53

a

100

100-5

80
60
40

b

20
0

0

c

c

d

e

1
2
3
4
5 （mm）
Thickness of dentin
（mm）

a
300-5

80
60
40

b

20
0

0

c

d

e

f

1
2
3
4
5 （mm）
Thickness of dentin（mm）
Fig. 3 Transmittance of Nd: YAG laser for different dentin thicknesses
Different letters indicate statistically significant differences at p＜0.05.

was 26.5±1.7％ for the DT1 group, 20.5±1.2％ for theUnder all the irradiation conditions, the E（＋）group

DT2 group, 10.9±0.6％ for the DT3 group, 11.5±1.1％

showed a significant attenuation of the transmittancefor the DT4 group, and 8.6±0.4％ for the DT5 group.

compared to the E（－）group.

Transmittance was significantly attenuated withincreasing dentin disc thickness except for the DT3 andDT4 groups.
The inhibition ratio under irradiation condition 100‒5
was 22.8±1.8％ for the DT1 group, 16.9±0.5％ for the

2 ．Experiment 2: Review of dentinal tubule sealing abilities
Table 3 shows the results of measuring the permeability inhibition ratio after laser irradiation under eachcondition.

DT2 group, 16.9±2.3％ for the DT3 group, 11.9±1.4％

The permeability inhibition ratio under irradiation

for the DT4 group, and 7.3±0.4％ for the DT5 group.

condition 50‒50 was 24.4±3.2％ in the 10 s group, 44.0

Transmittance was significantly attenuated with

±6.3％ in the 20 s group, and 55.4±3.6％ in the 30 sincreasing dentin disc specimen thickness except forgroup. The permeability inhibition ratio of the 30 s

the DT2 and DT3 groups.

group was significantly higher than that of the 10 s and

The inhibition ratio under irradiation condition 300‒5
was 23.2±2.2％ for the DT1 group, 18.8±1.6％ for theDT2 group, 13.9±0.9％ for the DT3 group, 11.1±0.7％

20 s groups, while that of the 20 s group was significantly higher than that of the 10 s group.
The permeability inhibition ratio under irradiation

for the DT4 group, and 7.8±0.5％ for the DT5 group. In

condition 100‒20 was 26.8±10.0％ in the 10 s group,

all groups, transmittance was significantly attenuated

43.1±7.3％ in the 20 s group, and 53.2±10.1％ in the 30

with increasing dentin disc specimen thickness.

s group. The permeability inhibition ratio of the 20 s

Under all the irradiation conditions, the transmit-and 30 s groups was significantly higher than that oftance of the DT1 group was significantly lower than

the 10 s group, and no significant difference was foundthat of the DT0 group, with greater attenuation.

in the permeability inhibition ratios of the 20 s and 30 s

Figure 4 shows the results of transmittance of thelaser in combination with TiO2 solution.

groups.
The permeability inhibition ratio under irradiation

ODEP Vol. 2, No. 1
50-10

80
60
40
20
0

b
E
（-）

E（＋）

a

100

300-5

80
60
40
20

b

0

E（-）

TiO2 solution
Fig. 4100-5Transmittance of laser（％）

100aTransmittance of laser（％）

Transmittance of laser（％）

54

E
（＋）

TiO2 solution

100

a

80
60
40
20
0

b
E
（-）

E
（＋）

TiO2 solution

Transmittance of Nd: YAG laser in combination with TiO2 solution for the dentin
Different letters indicate statistically significant differences at p＜0.05.

Table 3

Measurement of the dentin permeability inhibition ratio in Experiment 2
Dentin permeability inhibition ratio（％）
10 s
20 s
30 s

Irradiation time
50‒50
Irradiation condition

100‒20100‒99300‒2024.4±3.2A,a26.8±10.049.3±6.9A,aA,b27.5±12.7A,a44.0±6.3B,a43.1±7.3

B,a55.4±3.6C,a53.2±10.1B,a43.1±15.2

A,a40.5±9.5A,a43.2±10.9

A,a41.2±8.4A,aThe results are shown as the mean±SD. Different lowercase letters
indicate statistically significant differences at p＜0.05 for the same irradiation time. Different uppercase letters indicate statistically significant
differences at p＜0.05 for the same irradiation conditions.

condition 100‒99 was 49.3±6.9％ in the 10 s group, 43.1

Figure 5 shows the results of SEM observations of±15.2％ in the 20 s group, and 40.5±9.5％ in the 30 s

the dentin disc specimen surfaces under each irradia-

group. There was no significant difference in the per-tion condition. The SEM images of each group indicatedmeability inhibition ratio among the 10 s, 20 s, and 30 sthat dentinal tubules were sealed by cohesive struc-groups.

tures. Under each irradiation condition, the longer the

The permeability inhibition ratio under irradiation

irradiation time, the more the dentinal tubules werecondition 300‒20 was 27.5±12.7％ in the 10 s group,

sealed by the cohesive structures and the layers

43.2±10.9％ in the 20 s group, and 41.2±8.4％ in theformed by the reactants of TiO 2 solution and laser

30 s group. There was no significant difference in the（white arrow）. In the 100‒99 group（20 s and 30 s）andpermeability inhibition ratio among the 10 s, 20 s, andthe 300‒20 group（20 s and 30 s）, the dentin surface was

30 s groups.

found to be melted in some areas（black arrow）.

The permeability inhibition ratio of the 100‒99 groupwas significantly higher than that of the 50‒50, 100‒20,

Discussion

and 300‒20 groups during the 10 s irradiation time. The
50‒50, 100‒20, and 300‒20 groups showed similar per-Dentin hypersensitivity is treated by a methodmeability inhibition ratios with no significant difference.

（HLLT）using a surface-absorbing laser（Er: YAG

The permeability inhibition ratios of all the groups

laser21,22）or CO2 laser20,24））
to seal the dentinal tubules by

were not significantly different and were similar at the

denaturing the proteins in the tubules and to close theirradiation times of 20 s and 30 s, respectively.

exposed dentinal tubule openings, thereby suppressingDec, 2022

Sealability of Dentinal Tubules Irradiated by Nd: YAG Laser with TiO2

No
irradiation

10 s

20 s

30 s

50-50

100-20100-99300-20Fig. 5 SEM observations of the dentin specimen surfaces under each irradiation condition
The dentinal tubules were sealed by the cohesive structures and the layers formed by thereactants of TiO2 solution and Nd: YAG laser（white arrow）. In the 100‒99 group（20 s and 30 s）
and the 300‒20 group（20 s and 30 s）, the dentin surface was found to be melted in some areas（black arrow）.

55

ODEP Vol. 2, No. 1

56

irritation to the fluid in the dentinal tubules27,39）. In addi-

absorbs laser light efficiently, the transmittance of thetion, the LLLT method is reported to reduce or allevi-

laser may have been attenuated due to the use of TiO2

ate pain by inhibiting the activity of Aδ or C fibers ofsolution.

peripheral nerves in the pulp using a GaAlAs semiconductor laser

23）

or Nd: YAG laser

19）

which are tissue

penetrating lasers40‒43）.

In this study, dentin disc specimens were preparedby forming from the occlusal surface based on themethod of Zennyu etc.27） to produce the specimen size

Dentin is a semi-permeable material with respect toand the open dentinal tubules. The results of the per-the Nd: YAG laser light used in this experiment 44）.

meability inhibition ratio after laser irradiation com-However, excessive laser irradiation can cause the laser

bined with TiO2 solution confirmed permeability inhibi-

light to reach the inside of the dentin, heating and pos-tion under all conditions. SEM images showed that the45）

sibly damaging the dental pulp . In this experiment,

open dentinal tubules on the dentin surface were sealed

the transmittance of Nd: YAG laser was attenuated toby the cohesive structures under all conditions. It has

about 25％ at a dentin disc thickness of 1.0 mm, regard-

also been reported that when TiO2 solution was irradi-

less of the irradiation conditions. The transmittance of

ated with Nd: YAG laser, the temperature increased

Nd: YAG laser decreased more with increasing thick-due to the heat generated with the absorption of laser

ness of the dentin disc. There are also reports indicat-

light by TiO2, rather than due to the effect of photoca-

ing that the transmittance of the laser at a dentin thick-talysis caused by the irradiation25,51）. This suggests thatness of 1.0 mm is about 25 to 45％46,47）. Dentin is highly

the combination of Nd: YAG laser and TiO2 solution

permeable to Nd: YAG laser due to the low absorption

caused thermal denaturation of proteins and other sub-rate of hydroxyapatite, which is a component of dentin.

stances in the dentinal tubular fluid, thereby sealing the

Therefore, the irradiation energy can be attenuated bydentinal tubules. The significantly higher permeability

more than 80％ if there is 3 mm or more of healthy

inhibition ratio in the 100‒99 group under the 10 s irra-

dentin between the pulp and the laser. This means thatdiation condition may be due to the effect of the higher

even if there is 5 mm of healthy dentin, the energy can-energy density than in the other groups. It is assumed

48）

that the laser irradiated at a high energy density

When TiO2 was used as a photoreactive agent during

reacted efficiently with the TiO2 solution to generate

Nd: YAG laser irradiation, the transmittance of thehigh thermal energy. Therefore, temperatures above

laser was greatly attenuated to less than 5％ regardless

60℃52） at which the proteins begin to denature andof the laser irradiation conditions. The photoreactive

coagulate occurred, resulting in good dentinal tubule

agent TiO2 has anatase and rutile crystal structures.

sealing abilities after a short irradiation time. However,TiO2 has two applications: it is used as a white pigment

the permeability inhibition ratio increased with increase

with suppressed photocatalysis, and it is also used forof the irradiation time for the irradiation conditions of

not be completely absorbed .

49,50）. When50‒50 and 100‒20, and there was no significant differ-

photocatalytic activity is not required, the rutile typeence in the permeability inhibition ratio of each groupwith low activity is used, whereas the anatase type

for the irradiation conditions of 20 s and 30 s. Thesewith high activity is used when photocatalytic activity

results suggest that the reaction with the TiO2 solution

is required. TiO2 with the rutile type crystalline struc-

was accelerated and sufficient thermal energy was gen-ture was used in this experiment, as it is almost com-erated by prolonged irradiation time, even at low

pletely free of photocatalysis. In addition, the TiO2 pow-

energy densities. Therefore, the same level of dentinal

photocatalysis, each with different properties

der surface was coated with inert alumina（Al2O3）totubule sealing ability as that obtained by laser irradia-

suppress chemical reactivity, including photocatalysis.

tion at high energy densities may have been obtained.

TiO2 produces reactive oxygen species through photo-

However, the permeability inhibition ratio did notcatalysis, but the rutile type requires energy fromincrease with increasing irradiation time under the irra-

50）. Thus, it isdiation conditions of 100‒99 and 300‒20. SEM images ofunlikely that the 1,064 nm Nd: YAG laser produces

the dentin surface showed images of dentin melting.

reactive oxygen species, and the possibility of photocat-

There are reports that dentin denaturation occurs due

alytic reaction is extremely low. However, since TiO2

to the generation and accumulation of higher thermal

short-wavelength light below 413 nm

Dec, 2022

Sealability of Dentinal Tubules Irradiated by Nd: YAG Laser with TiO2

57

energy at higher energy densities 9,10）. The thermalenergy that occurred by high energy densities under
the irradiation conditions of 100‒99 and 300‒20 was
seemingly too high to seal the dentinal tubules by denaturation and coagulation of the proteins. Dentin degeneration is considered to influence the sealing ability ofdentinal tubules. Hence, we concluded that the permeability inhibition ratio may have not increased in proportion to the increase of the energy density. Therefore,
Nd: YAG laser irradiation combined with TiO2 solution
is effective for sealing dentinal tubules. However, inorder to obtain good dentinal tubule sealing without
dentin degeneration, care must be taken with the highenergy density of the laser.
Furthermore, by investigating the flow rate of the
TiO2 solution, there is a possibility that Nd: YAG laser
could provide a less invasive and more effective treatment method for dentin hypersensitivity by demonstrating the effect of HLLT in addition to the original
LLLT effect of Nd: YAG laser.

Conclusions
The effects of dentin thickness or TiO2, a photoreactive agent, on Nd: YAG laser penetration into dentin,
and the effect of Nd: YAG laser irradiation with TiO2
on the sealing of dentinal tubules were examined, andthe following results were obtained.
1．The transmittance of Nd: YAG laser through dentin decreased with increasing dentin thickness.
2．The transmittance of Nd: YAG laser through dentin decreased significantly when TiO2 was used in combination.
3．When Nd: YAG laser was irradiated with TiO2,
the sealing abilities of dentinal tubules improved. At
100μs‒100 mJ‒99 pps, a high dentin permeability inhibition effect was observed after a short irradiation time.
At 50μs‒50 mJ‒50 pps and 100μs‒100 mJ‒20 pps, thedentin permeability inhibition ratio increased withincreasing irradiation time.
Conflict of Interest
The authors declare no conflict of interest in this study orreport.

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ODEP Vol. 2, No. 1

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Original Article

ODEP 2（1）：60～71，2022

Application of Intraoral Scanners in Dental Health Guidance―Quantification and Visualization of Marginal Gingival Changes―
Akina TANI, Akane OMORI, Shizuo HIGUCHI＊ and Kazutoshi KAKIMOTO＊
Osaka Dental University Graduate School of Health Sciences＊

Department of Oral Health Engineering, Faculty of Health Sciences, Osaka Dental University

Abstract
Introduction: We have been studying the effectiveness of the intraoral scanner（IOS）as a tool for dental
health guidance. We have confirmed the possibility of scanning the gingiva with high precision and of capturing gingival changes quantitatively and visually by aligning the scan data. In the present study, we investigated whether the shape changes of marginal gingiva generated experimentally by inserting a gingival
retraction cord into the gingival sulcus could be quantified and visualized by using IOS recording. In addition,
we also studied the improvement of accuracy by alignment method. In other words, the objective of thisstudy was to quantify and visualize the clinical shape changes of the gingiva with high precision by IOS, andto connect them to clinical applications.
Subjects and methods: The subjects were three faculty members of the Faculty of Health Sciences, Osaka
Dental University. First, the oral cavity was scanned three times repeatedly. Then, gingival retraction cords
were inserted into the gingival sulcus of four teeth, two in the maxilla and two in the mandible, for eachstudy subject. The data was saved for IOS and as exported data in Standard Triangulated Language（STL）
format. The STL data before and after gingival retraction were compared using three-dimensional inspection
software.
Results: In the prealignment, which is the aligning of the scanned whole before and after gingival retraction, the deviation on the tooth surface was within ±0.5 mm in the interquartile range（IQR）for most of themeasurement points. The deviation on the gingiva was slightly more variable than that on the tooth surface.
For the local best-fit alignment on all tooth surfaces, the deviation on the tooth surface was smaller and lessvaried than that for the prealignment. On the other hand, the deviation at the gingiva in the local best-fit
alignment on all tooth surfaces was larger and more varied than that in the prealignment. As for marginal
gingival changes, 97.7％ had bulging marginal gingiva and 89.4％ had depressed gingival sulcus in the prealignment. In the local best-fit alignment on all tooth surfaces, 98.1％ had distended marginal gingiva and89.4％ had depressed gingival sulcus. In the local best-fit alignment on the gingival retracted tooth surface,
97.7％ had bulging marginal gingiva and 88.4％ had depressed gingival sulcus.
Conclusions: The following conclusions were drawn from these results: 1. There was little difference inquantification or visualization between prealignment, which optimally aligned both tooth and gingival surfaces, and alignment, which optimally aligned all tooth surfaces in the dentition. 2. Although there is an error
in the scanning of the oral cavity by IOS, it is precise enough to be used for dental health guidance. 3. Intraoral scans by IOS allow quantification and visualization of gingival changes.
Key words: intraoral scanner, dental health guidance, gingival change

Corresponding author: Akina TANI, Osaka Dental University Graduate School of Health Sciences, 1‒4‒4, Makinohonmachi,
Hirakata, Osaka 573‒1144, Japan
TEL: ＋81‒72‒856‒9985, FAX: ＋81‒72‒856‒2111, E-mail: tani-a@cc.osaka-dent.ac.jp
Received for Publication: September 15, 2022/Accepted for Publication: November 7, 2022
DOI: 10.11471/odep.2022‒008

Dec, 2022

Intraoral Scanners in Dental Health Guidance: Quantification and Visualization

61

previous study2）, the maximum standard deviation of
the distance between measurement points was 0.30 mm

Introduction

and its standard error was 0.12 mm. In another study3）,
Currently, intraoral scanners（IOS）are mainly used

it was possible to quantify and visualize a film with ain the fabrication of prosthetic and orthodontic devices.

thickness of 0.11 mm applied to the gingiva. Based on

We have been investigating the possibility of using thisthe above, we considered that the objectives of thetechnology not only for the fabrication of prosthetic

study could be achieved with three subjects based on

devices, but also for a wide range of dental treatments.

the thickness of the retraction cords. Subjects excluded

In particular, we expect that intraoral scanned records

were those with three or more consecutive missing

captured by IOS will replace study casts and intraoral

teeth, those who had extreme difficulty inserting gingi-

photographs in dental health guidance.

val retraction cords into the gingival sulcus due to mar-

The color tone cannot be confirmed on study casts.

ginal gingival tension or severe pain during gingival

In addition, intraoral photographs are not three-dimen-

retraction, and those who did not give their consent.

sional. Scanning and recording by IOS overcomes both

The selection criteria were those who understood theof these issues and is considered to be of high value.

study, agreed to participate in the study, and had no1）

Furthermore, the operation time is short , and lessevidence of periodontal disease from an examination byinvasive for the patient. There is also no problem ofa dental hygienist（not a JSP Dental Hygienist）with atspace for storing the casts.

least 15 years of practical experience. The subjects

Therefore, we have been studying the usefulness of
IOS for dental health guidance

2,3）. First, the accuracy ofwere one male and two females with a mean age of
48.0 years.

recording a complete denture was confirmed assuming

2 ．Scanning methods

an oral cavity, and the possibility of quantification and

TRIOS 3（3shape, Copenhagen, Denmark）was used2）

visualization of shape change was examined . In addi-

as the IOS. First, the oral cavity was scanned in tripli-

tion, we applied film-type stomatitis patches to the gin-cate. Following the procedures of the supplied measure-

gival region in the actual oral cavity to determine thement software, the intraoral cavity was scanned first inaccuracy of the recording against changes in periodon-

the mandible, then in the maxilla, and finally in the3）

tal tissue caused by IOS .

occlusion. Then, a total of four teeth were selected, two

In the previous study, the shape change was identi-

each in the maxilla and mandible, in which it was easy

fied due to changes caused by the application of theto insert the retraction cords in order to avoid subject

film agent, and the gingiva was unchanged. Therefore,

fatigue and allow a longer time interval before andin this study, the shape of the marginal gingiva wasafter retraction. Table 1 shows gingival retracted teeth

experimentally changed by inserting a gingival retrac-

and their probing depths in each subject. Bleeding attion cord into the gingival sulcus. We then confirmed

the time of insertion of the gingival retraction cord may

the quantification and visualization of slight changes inaffect IOS recording. Therefore, teeth that did not

the gingiva and examined the extent to which the accu-

bleed during probing were selected as test teeth. Gingi-

racy of quantification and visualization could be

val retraction cords（Ultrapak cord, Ultradent Products,

improved by changing the alignment method. The pur-South Jordan, UT, USA）were inserted into the gingival

pose of this study was to quantify and visualize thesulcus of these teeth. The cords used were #000（0.89

clinical shape changes in the gingiva with high accu-

mm diameter）or #00（1.04 mm diameter）. The oralracy by IOS, and to connect them to clinical applica-

cavity was then scanned three times in the same man-tions.

ner as before gingival retraction.
3 ．Evaluation methods

Subjects and Methods

The scanning data were stored as TRIOS 3-specificdata and exported to STL format for storage. The STL

1 ．Subjects

data before and after gingival retraction were thenThe subjects were three faculty members of the Fac-

compared using three-dimensional inspection software

ulty of Health Sciences, Osaka Dental University. In a

（GOM Inspect 2016, GOM, Braunschweig, Germany）inODEP Vol. 2, No. 1

62
Table 1
Subject

Gingival retracted teeth and their probing depths in each subject

Tooth

Probing depth（mm）
DistalMaxillaRight central
incisor222Palatal222DistalMandible

Right ﬁrst
molar

Left central
incisor

Lingual3

23

Buccal

3

2

3

2

1

2

Palatal2

11DistalMandible

Right second
premolar

Lingual1

11Buccal1

1

3

Right central
incisor222Palatal222Subject C

DistalMandible

Right canine

2

1

2

Palatal222

MesialLeft second
molar

3

3

3

Buccal3

2

3

MesialLeft lateralincisor2

2

2

Labial222DistalLabial112Palatal1

11

MesialLeft second
premolar

DistalLingual2

12

Buccal3

1

2

MesialLeft ﬁrst
molar

DistalBuccal323Palatal222

MesialLingualDistalLingualMesialLabialDistalLabial

MesialDistalMaxillaLeft central
incisorDistalLabialSubject B

Mesial

Mesial

MesialMaxilla

Probing depth（mm）

MesialLabialSubject A

Tooth

MesialLeft ﬁrst
premolar

DistalLingual222Buccal2

2

2

No bleeding on probing was observed on any of the teeth.

the following way.
The data before retraction and after retraction wereread as CAD data（nominal elements）and mesh data
（actual elements）, respectively. The measurement
points were then obtained on the surface of the meshdata, i. e., the data after gingival retraction. An exampleof the measurement points on the labial and buccal sideof the mandible is shown in Figure 1. The measurement points were set with reference to the position of
periodontal pocket measurement for future application
to oral health guidance. A total of six measurement

Fig. 1

Example of the measurement points on thelabial and buccal side of the mandible

points were set on the labial, buccal, and lingual surfaces of each tooth: mesial, median, and distal, and six

at the measurement points were calculated for the

points on the keratinized gingival area corresponding toaligned images. Then, alignment was also performed on

the measurement points on the tooth surface. The CAD

all tooth surfaces of the dentition, and the deviation atand mesh data were then prealigned. Prealignment iseach measurement point was obtained. In addition,

the initial alignment, in which both the tooth surfacealignment was also performed on the tooth surfaces ofand gingival surface are aligned for best fit. Deviations

gingival retracted teeth. One scanning data before gin-Dec, 2022
Table 2Intraoral Scanners in Dental Health Guidance: Quantification and Visualization

63

Rating criteria of gingival changes around gingivally retracted teeth by viewing surface comparison
images before and after gingival retraction in surface comparison images
Degree of marginal gingival bulge

Degree of gingival sulcus depression

Color indicating the deviation

Determination

Color indicating the deviation

Determination

Only green, light blue or blue
Yellow unclearly viewed
Yellow clearly viewed
Red unclearly viewed
Red clearly viewed

0
1
2
3
4

Only green, yellow or red
Light blue unclearly viewed
Light blue clearly viewed
Blue unclearly viewed
Blue clearly viewed

0
1
2
3
4

Blue is approximately －0.16 mm or less, light blue －0.15 mm to －0.06 mm, green －0.05 mm to ＋0.05 mm,yellow ＋0.06 mm to ＋0.15 mm, and red ＋0.16 mm or more.

gival retraction was aligned with three scanning data

and the results were subjected to Bonferroni correc-

after retraction, respectively. Since three scans weretion.

also made before gingival retraction, the number of

5 ．Ethical approvalaligned combinations was nine multiplied by three

This study was approved by the Osaka Dental Uni-times after retraction by three times before retraction

versity Medical Ethics Committee（Approval No.

in one subject.

111173-0）. The materials and equipment used have

Regarding color, the criteria for evaluating the visible
changes in color on comparative images of the marginal

been approved by the Ministry of Health, Labour andWelfare.

gingiva of the gingival retracted teeth are shown in
Table 2. The marginal gingiva was evaluated on a
5-point scale, with“4”for a bulge of ＋0.16 mm or moreindicated in red and“0”for almost no bulge from －0.05

Results
1 ．Surface comparison in areas with no change

mm to ＋0.05 mm indicated in green. The gingival sul-

Examples of comparative images of intraoral records

cus was evaluated on a 5-point scale, with“4”for a

before and after gingival retraction scanned using IOS

depression of －0.16 mm or less indicated in blue andby prealignment, local best-fit alignment on all tooth“0”for almost no depression from －0.05 mm to ＋0.05

surfaces, and local best-fit alignment on the gingival

mm indicated in green.

retracted tooth surface are shown in Figure 2. Three

4 ．Statistical analysis

scans were taken before and after each gingival retrac-

Statistical analysis was performed as follows. The

tion, and the time interval recorded before and after

absolute value of the deviation was used as the error,

gingival retraction was about 2 hours at most. There-

and the influence of the site and alignment method onfore, there was no shape change except for the mar-the error was analyzed by two-way ANOVA withginal gingiva of the gingival retracted tooth and theregion（tooth surface and gingiva）and alignment

movable portion of the gingiva. However, there was no

method（prealignment and local best-fit alignment on all

record of a perfect match before and after gingival

tooth surfaces）as factors. The effect size was also cal-indentation. Visual comparison of the prealigned andculated. In addition, the differences in respective errors

aligned images on all tooth surfaces showed little differ-

between the tooth and gingival surfaces due to the dif-ence in the comparative images before and after gingi-

ferent alignment methods were analyzed with a paired

val retraction in any of the subjects.

means t-test.

On the other hand, morphological changes were

For the evaluation of marginal gingival changes,

observed in the marginal gingiva of the gingival

Friedman’s test was performed using three different

retracted teeth in most of the images. In the mandiblealignment methods as factors: prealignment, local best-of the subject in Figure 2, gingival retraction was per-fit alignment on all tooth surfaces, and alignment on theformed on the right canine and left first premolar. In

gingival retracted tooth surface. In addition, Wilcoxon’s

Figure 2, changes in the marginal gingiva of the gingi-

signed-rank test was performed for each combination,

val retracted teeth were observed in both the prealign-ODEP Vol. 2, No. 1

64← 0.25 mmteeth, especially on the contralateral side.
Figure 3 shows examples of comparative images
around the gingival retracted maxillary central incisorsby alignment of the three methods. The area with alarge deviation from the brown band visible in the mid-（a）

← －0.25 mmline is the gingival retraction cords. The marginal gingiva of the gingival retracted tooth is indicated in red,
indicating that the gingiva was augmented by approxi-← 0.25 mm

mately 0.2 mm. There was less gingival augmentationon the lingual side than on the labial side. In addition,
the gingival sulcus indicated in blue shows that it wasdepressed after gingival retraction. The deviation values varied slightly depending on the alignment method.

（b）← －0.25 mm

Figure 4 shows box-and-whisker plots of the distribution of surface deviation compared by the prealignmentof the IOS records before and after gingival retraction.

← 0.25 mm

The measurement points of the gingiva were set at theattached gingiva region. The number of data at eachmeasurement point varied slightly, from 105 to 162,
because some records had congenitally missing teeth ormeasurement points were not in the scanned range.

（c）← －0.25 mm

Comparative images of records before andafter gingival retraction scanned using IOS
by alignment of the three methods（a）Prealignment,（b）Local best-fit alignment on all
tooth surfaces,（c）Local best-fit alignment on thegingival retracted tooth（in this figure, the mandibular right canine）surface.
The redder the deviation, the more positive thedeviation, and the bluer the deviation, the more negative the deviation. A positive deviation indicates thatthe surface after gingival retraction is outside of thesurface before retraction. A negative deviation indicates that the surface after gingival retraction is
inside of the surface before retraction.
Fig. 2

Deviations of most measurement sites on the tooth surface were within ±0.5 mm in the interquartile range（IQR）. On the other hand, there were a few outlierswith deviations exceeding 0.20 mm. The deviation on
the gingiva was slightly more variable than that on thetooth surface.
Figure 5 is a graph similar to Figure 4 with local
best-fit alignment on all tooth surfaces. The deviationon the tooth surface was smaller than the prealignment
and the variation was also smaller. On the other hand,the deviation in the gingival region was larger than theprealignment and the variability was also larger.
ANOVA results showed that both the factors of site
（tooth surface and gingiva）and alignment method（prealignment and local best-fit alignment on all tooth surfaces）and the interaction of these two factors had significant effects（p＜0.01）. However, the effect sizes ofment and the local best-fit alignment on comparison

the alignment methods were small: 0.080 for the tooth

images of all tooth surfaces. Furthermore, when align-

surface and －0.176 for the gingiva.

ment was performed on the surface of the gingival

Figure 6 compares the absolute values of deviations

retracted tooth―the right canine tooth in Figure 2

by alignment method. When the prealignments were（c）―the deviation of the canine tooth surface was

replaced by local best-fit alignment on all tooth sur-

reduced and the marginal gingival changes appeared

faces, the error decreased from 0.036 mm to 0.034 mm

slightly different from the prealignment and local best-on the tooth surface and increased from 0.044 mm tofit alignment on all tooth surfaces. On the other hand,

0.052 mm on the gingiva. The results of the paired

deviations were larger in areas other than the canine

means t-test showed that the differences in errors wereDec, 2022

Intraoral Scanners in Dental Health Guidance: Quantification and Visualization

65

Gingival retraction cords← 0.25 mm＋0.21 mm－0.01 mm＋0.01 mm＋0.13 mm（a）

← －0.25 mm

Gingival retraction cords← 0.25 mm＋0.21 mm－0.02 mm＋0.01 mm＋0.13 mm

← －0.25 mm（b）
Gingival retraction cords← 0.25 mm＋0.20 mm－0.01 mm－0.00 mm＋0.13 mm

← －0.25 mm（c）Fig. 3 Comparative images around the gingival retracted maxillary central incisor by alignment of the threemethods（a）Prealignment,（b）Local best-fit alignment on all tooth surfaces,（c）Alignment on the surface of the left
central incisor.
The color display is the same as in Figure 2.

all significant at the 1％ level of significance. On thein Table 2 were tabulated by tooth alignment method,other hand, the mean difference was 0.002 mm for the211（97.7％）had bulging marginal gingiva and 193

tooth surface and －0.008 mm for the gingiva, which（89.4％）had depressed gingival sulcus in the prealign-

were very small. The standard deviations were 0.030

ment. In the local best-fit alignment on all tooth sur-mm for both prealignment and local best-fit alignment

faces, 212（98.1％）had distended marginal gingiva andon all tooth surfaces on the tooth surface, and 0.044 mm

193（89.4％）had depressed gingival sulcus. In the local

for prealignment and 0.052 mm for local best-fit align-

best-fit alignment on the gingival retracted tooth sur-ment on all tooth surfaces on the gingiva.

face, 211（97.7％）had bulging marginal gingiva and 191

2 ．Marginal gingival changes in gingival retracted
teeth
Table 3 shows the results of evaluating the changes（88.4％）had depressed gingival sulcus.
Most of the teeth with gingival retraction were anterior teeth in the maxilla and molars in the mandible.

in the marginal gingiva of the gingival retracted teeth

Figure 7 compares the percentage of the evaluation

by looking at the comparative images before and after

results of the changes in the marginal gingiva before

gingival retraction. Of the 216 sites for which the data

and after gingival retraction with the different align-ODEP Vol. 2, No. 1
0.30

0.20

0.20

0.10
0

－0.10
－0.20
－0.30

Surface deviation, mm

Surface deviation, mm

0.30

0.10
0
－0.10
－0.20
－0.30

7 6 5 4 3 2 1 1 2 3 4 5 6 717 16 15 14 13 12 11 21 22 23 24 25 26 27
Dentition
（a）

0.30

0.30

0.20

0.20

0.10
0
－0.10
－0.20
－0.30

7 6 5 4 3 2 1 1 2 3 4 5 6 717 16 15 14 13 12 11 21 22 23 24 25 26 27
Teeth near the gingiva（c）

7 6 5 4 3 2 1 1 2 3 4 5 6 747 46 45 44 43 42 41 31 32 33 34 35 36 37
Dentition（b）

Surface deviation, mm

Surface deviation, mm

66

0.10
0
－0.10
－0.20
－0.30

7 6 5 4 3 2 1 1 2 3 4 5 6 747 46 45 44 43 42 41 31 32 33 34 35 36 37
Teeth near the gingiva（d）Fig. 4 Box-and-whisker plots showing surface in prealignment（a）Maxillary tooth surface,（b）Mandibular tooth surface,（c）Maxillary gingival surface,（d）Mandibular gingival surface.

ment methods. About half of the patients had the high-cance level was set at 10％, local best-fit alignment on

est rating of 4 for marginal gingival bulge. The ratings

the gingival retracted tooth surface was rated lowerof 4 increased in the order of prealignment, local best-

than prealignment.

fit alignment on all tooth surfaces, and local best-fit
alignment on the gingival retracted tooth surface. In

Discussion

contrast, in the gingival sulcus, the highest rating of 4
accounted for approximately 30％. In addition, the rat-

1 ．Scanning precisionings of 4 decreased slightly in the order of prealign-

There have been many studies on the trueness andment, local best-fit alignment on all tooth surfaces, andprecision of teeth and implants scanned by IOS4‒6）,

local best-fit alignment on the gingival retracted tooth

which have shown that scanning with IOS is clinicallysurface.

acceptable with excellent trueness and precision. In

Bonferroni correction of the Wilcoxon signed-rank

addition, there have recently been several studies on

test results for the alignment method combinations

the use of IOS for gingival examination7‒9）. However,showed that for marginal gingival bulge, local best-fit

there have been few studies on the accuracy of scan-alignment on all tooth surfaces and local best-fit align-ning of gingiva10,11）. Therefore, we measured the error

ment on the gingival retracted tooth surface wereof scanning in the gingival region of the same denture

rated higher than prealignment（p＜0.05）. No combina-

six times using a complete denture2）and found that thetion was significant at the 5％ level of significance forrecording precision was high even in the gingival

the depression of the gingival sulcus. When the signifi-

region.

Intraoral Scanners in Dental Health Guidance: Quantification and Visualization

0.30

0.30

0.20

0.20

Surface deviation, mm

Surface deviation, mm

Dec, 2022

0.10
0
－0.10
－0.20
－0.30

0.30

0
－0.10
－0.20

0.30

7 6 5 4 3 2 1 1 2 3 4 5 6 747 46 45 44 43 42 41 31 32 33 34 35 36 37
Dentition
（b）

0.20

Surface deviation, mm

0.20

Surface deviation, mm

0.10

－0.30

7 6 5 4 3 2 1 1 2 3 4 5 6 717 16 15 14 13 12 11 21 22 23 24 25 26 27
Dentition
（a）

67

0.10
0
－0.10
－0.20
－0.30

7 6 5 4 3 2 1 1 2 3 4 5 6 717 16 15 14 13 12 11 21 22 23 24 25 26 27
Teeth near the gingiva
（c）

0.10
0
－0.10
－0.20
－0.30

7 6 5 4 3 2 1 1 2 3 4 5 6 747 46 45 44 43 42 41 31 32 33 34 35 36 37
Teeth near the gingiva（d）Fig. 5 Box-and-whisker plots showing surface deviations in alignment on the surface of the dentition（a）Maxillary tooth surface,（b）Mandibular tooth surface,（c）Maxillary gingival surface,（d）Mandibular gingival surface.
0.06
Deviation, mm

0.05

Bar: 95% conﬁdence interval

precision may be reduced due to the presence of saliva
and other intervening substances in the oral cavity.
Therefore, in this study, the results of multiple intraoral

0.04

scans were aligned and deviations were displayed to

0.03

confirm the precision.

0.02

As shown in the example in Figure 2, even in the

0.01

same oral cavity, areas other than the marginal gingiva

0Fig. 6of the right canine and left first premolar with gingival
Gingiva
Tooth
Prealignment
Alignment on the surface of the dentition

retraction were also found to be other than green, indi-Comparison of absolute values of deviation
between prealignment and local best-fit alignment on all tooth surfaces

shows that the deviations of each region were in thecating less deviation. In other words, no records withperfectly matched scan data were obtained. Figure 4
IQR, except for the gingival region of the mandibularleft second molar, when prealigning was performed, i. e.,
the two entire 3D data sets were analyzed and aligned

However, in intraoral scanning, the scanned object

at the best-fitting position. That is, 50％ of the measure-cannot be taken out of the mouth like a complete den-ments were within ±0.10 mm. When not including out-ture, so it is impossible to scan while holding the objectliers, which are values more than 1.5 quadrants apart,in the hand and moving it. In addition, the recording

all values were within ±0.20 mm, except for the maxil-

ODEP Vol. 2, No. 1

68

Results of rating of gingival changes around gingivally retracted teeth by viewing surface comparison images before

Table 3and after gingival retraction
Marginal gingival region
Position

Anterior
tooth

MaxillaPosterior
tooth

Anterior
tooth

Mandible

Posterior
tooth

Alignment method

Gingival sulcus region

Side

4

3

2

1

0

Total

4

3

2

1

0

Total

Prealignment

LabialPalatal

26
15

10
6

8
10

1
14

0
0

45
45

31
14

3
9

5
12

5
9

1
1

45
45

On the surface of thedentition

LabialPalatal

23
18

11
3

10
13

1
11

0
0

45
45

30
12

5
6

5
14

4
12

1
1

45
45

On the surface of thegingivally retracted tooth

LabialPalatal

30
19

5
3

8
10

0
13

2
0

45
45

32
15

2
3

6
10

4
16

1
1

45
45

Prealignment

BuccalPalatal

0
3

5
1

3
5

1
0

0
0

9
9

0
1

7
2

0
3

2
3

0
0

9
9

On the surface of thedentition

BuccalPalatal

3
6

3
2

3
1

0
0

0
0

9
9

0
0

7
3

0
0

2
6

0
0

9
9

On the surface of thegingivally retracted tooth

BuccalPalatal

2
6

5
2

2
1

0
0

0
0

9
9

0
0

6
1

0
1

2
7

1
0

9
9

Prealignment

LabialLingual

2
0

1
2

5
5

0
1

1
1

9
9

0
0

0
0

2
1

5
6

2
2

9
9

On the surface of thedentition

LabialLingual

2
0

2
4

4
4

0
1

1
0

9
9

0
0

1
0

1
1

5
5

2
3

9
9

On the surface of thegingivally retracted tooth

LabialLingual

0
3

0
2

7
4

2
0

0
0

9
9

0
0

0
0

0
0

7
5

2
4

9
9

Prealignment

BuccalLingual

21
29

8
3

13
6

3
4

0
3

45
45

15
8

3
6

2
5

18
16

7
10

45
45

On the surface of thedentition

BuccalLingual

22
30

6
2

11
6

6
4

0
3

45
45

12
9

5
5

2
7

19
15

7
9

45
45

On the surface of thegingivally retracted tooth

BuccalLingual

25
27

7
5

12
9

1
1

0
3

45
45

10
8

7
7

1
7

20
14

7
9

45
45

lary right second molar and mandibular left second

cavity were short, and the measurement point wasmolar gingival areas. Since deformation occurs during

included in the movable range of the gingiva.

alginate impression and plaster hardening even in the

Since it is the gingiva that is changed by dental

study cast, IOS scanning is considered to be accurate

health guidance, it seems reasonable to align theenough for dental health guidance.

scanned image on all tooth surfaces, i. e., on all remain-

On the other hand, outliers were also found. On theing tooth surfaces, rather than on the entire image.

tooth surfaces, more outliers were observed on the

Comparing（a）and（b）in Figure 2, visually little dif-anterior teeth than on the molars. This may be due toference was discernible between the two comparative

the fact that the incisal area of the anterior teeth is nar-images. The same was true for the other images exam-

rower than the occlusal surface of the molars, making it

ined in this study. Comparing the results of local best-fit

difficult to superimpose the images during scanning,alignment on all tooth surfaces（Figure 5）with theespecially on the labial and lingual sides. In the gingival

results of prealignment（Figure 4）, the IQR was slightly

region, outliers were also common in the molar region,

less on the tooth surfaces. In contrast, in the gingival

especially in the mandible. This may be because theregion, on the contrary, the IQR was slightly widened.

distance from the gingival margin of molars to the

The absolute value of the deviation shown in Figure 6, i.

mucobuccal fold and the distance to the floor of the orale., the error, was also smaller on the tooth surface andDec, 2022

Intraoral Scanners in Dental Health Guidance: Quantification and Visualization
Prealignment

4

3

On the surface of the dentition

4

On the surface of the gingivally retracted tooth

4

3

69

2

1

2

1 0

2

1 0

3

0

0％ 10％ 20％ 30％ 40％ 50％ 60％ 70％ 80％ 90％ 100％（a）Prealignment

4

On the surface of the dentition

4

3

On the surface of the gingivally retracted tooth

4

3

3

2

1

0

2

1

0

2

1

0

0％ 10％ 20％ 30％ 40％ 50％ 60％ 70％ 80％ 90％ 100％（b）Comparison of the percentage of the evaluation results of the changes in themarginal gingiva before and after gingival retraction with the differentalignment methods（a）Marginal gingival region,（b）Gingival sulcus region.

Fig. 7larger in the gingival region. These results are considered to be reasonable because of the alignment in thedentition tooth plane. On the other hand, although there

the future.
2 ．Quantification and visualization of gingivalchanges

was a significant difference in the mean values, theIn a previous study 3）, we investigated whether

effect of converting the prealigning to the local best-fit

changes caused by the film could be quantified andalignment on all tooth surfaces is considered to be small

visualized by aligning scan data before and after abecause the difference was small: the smallest unit of

0.11-mm-thick film was applied to the gingiva. As adeviation was 1/100 mm, and the standard deviation of

result, the thickness of the applied film could be accu-

each mean value was 0.030 to 0.052 mm, which is larger

rately quantified and visualized. On the other hand,than the difference in the mean values.

although the films were very thin, the boundaries were

In Figure 4（d）and Figure 5（d）
, the deviation was

clear, so it was necessary to evaluate the actual gingi-

larger for the posterior teeth in the mandible. In otherval changes in the actual oral cavity to see if they could

words, the gingiva was slightly swollen after the gingi-

be clarified. Therefore, in this study, the gingival

val retraction. The cause of this phenomenon will be

retraction cord was inserted into the gingival sulcus to

examined in the future.

alter the gingiva.

On the other hand, it is known that when a complete

As shown in Figures 2 and 3, the marginal gingiva ofarch is scanned by IOS, the trueness and precision are

gingival retracted teeth often had a bulge of approxi-

worse than when a partial arch is scanned12）. This ismately 0.2 mm or less. The diameter of the gingival

because IOS creates an overall image by gradually

retraction cord used was 0.89 mm for #000 and 1.04 mm

overlapping the scanned images and enlarging themfor #00, according to the attachment. Therefore, the13）

into an overall image . Therefore, in order to examine

bulge of the marginal gingiva was considerably smaller

the changes in the gingival area more accurately, it isthan the diameter of the gingival retraction cord. Thisconsidered necessary to align the data by block or by

may be due to the fact that the cords become thinnereach tooth surface. We would like to study this issue indue to compression of the receding gingiva and that the

ODEP Vol. 2, No. 1

70gingiva is also deformed by elasticity.

guidance can be expressed numerically. For example,

The deviation of the gingival bulge often slightly var-

the degree of gingival swelling can be expressed as aied depending on the method of aligning. In the exam-

numerical value based on breadth and height. Further-ple shown in Figure 3, the prealigning showed a labial

more, changes over time can also be expressed numeri-gingival bulge of 0.21 mm, whereas the local best-fit

cally. Visualization facilitates understanding for patientsalignment on the gingival retracted tooth surface

as well as dentists and dental hygienists.

showed a ridge of 0.20 mm. If aligning is performed

The ability to display changes in the oral cavity on a

only on the tooth surface of a tooth that has beenscale of 1/100 th of a millimeter may also allow clini-

retracted, the deviation will be large in the area away

cians and researchers to find changes that were previ-from the aligned tooth surface as shown in Figure （2 c）,

ously unknown.

so it should only be used to determine the gingiva of

Shape changes are determined by aligning the intra-

the tooth in question or its vicinity. Many of the gingi-

oral scanning record. Aligning methods are important

val retracted teeth also showed depressed changes into obtain changes accurately because of errors in

the gingival sulcus, indicated by the blue color. This

recording. We think that further study of alignment

may be due to the expansion of the gingival sulcus bymethods in clinical applications is needed.

gingival retraction.

Probing depth was the only method that could quan-

The visualization of gingival changes could be con-titatively represent changes in gingival shape in healthfirmed in approximately 98％ of the cases for marginal

guidance. With the use of IOS, detailed changes overgingival bulge and in approximately 89％ of the cases

time can be represented quantitatively and visually infor gingival sulcus depressions, as shown in Table 3,

an easy-to-understand manner. Therefore, in the future,

therefore it is considered to be sufficient for clinical use.

gingival changes in health guidance will be mainly

In Figure 7, the percentage of 4, which is the easiest forrecorded by IOS. On the other hand, probing will con-examining the marginal gingival bulge, increased in thetinue to be an important examination because probing

order of prealignment, alignment on the dentition, anddepth cannot be recorded.

alignment on the gingival surface of the compressed

In addition, areas that are difficult to observe on

tooth, indicating that alignment on the tooth surface

intraoral photographs, such as the lingual and palatal

makes the change easier to examine. However, the gin-surfaces, can be easily observed. It is predicted that thegival sulcus depression was rated less frequently thanclarity of images recorded by IOS will improve in thethe marginal gingival bulge, i. e., it was difficult tofuture. Therefore, IOS records are likely to be used as

examine. With respect to the depression of the gingival

an alternative to intraoral photographs.

sulcus, the alignment method had little effect on visual-In the future, we plan to study how gingival changes

ization. The cause may be that the gingival retraction

can be examined by using IOS in clinical dental healthcord is near the gingival margin or does not form a

guidance, and to verify whether dental health guidance

depression if it protrudes from the gingival sulcus, orusing IOS will be easier for patients to understand thanconversely, if the gingival retraction cord is inserted too

conventional guidance methods, and whether it will be

deeply into the gingival sulcus, the gingival sulcus

useful for dentists and dental hygienists who provide

closes. Another possible reason is that the light of IOS

such guidance.

does not reach the gingival sulcus sufficiently.
3 ．Clinical significance and future studies

Conclusions

Scanning with IOS provides a three-dimensional viewof the shape of the oral cavity. Furthermore, the results

The possibility of quantifying and visualizing gingivalof our previous studies and this study indicate thatchanges for the purpose of using IOS for oral healthchanges in oral shape can be quantitatively captured on

guidance was investigated.

the order of 1/100th of a millimeter. In addition, shapechanges could be visualized on a color display.
These indicate that gingival changes that could only
be expressed as“better”or“worse”in dental health

By repeated scanning of the oral cavity before andafter gingival retraction with IOS and evaluating theresults of aligning the obtained records, the following
conclusions were obtained:Dec, 2022

Intraoral Scanners in Dental Health Guidance: Quantification and Visualization

1. There was little difference in quantification or visualization between prealignment, which optimally aligned
both tooth and gingival surfaces, and alignment, which
optimally aligned all tooth surfaces in the dentition.
2. Although there is an error in the scanning of theoral cavity by IOS, it is precise enough to be used for
dental health guidance.
3. Intraoral scans by IOS allow quantification andvisualization of gingival changes.
Part of this study was presented at the 65th Spring Meeting of the Japanese Society of Periodontology（June 4, 2022,
Tokyo）.
Conflict of Interest
The authors declare no conflicts of interest associatedwith this manuscript.

References
1）Patzelt SB, Lamprinos C, Stampf S, Att W. The time
efficiency of intraoral scanners: An in vitro comparative
study. J Am Dent Assoc 2014; 145: 542 551.
2）Tani A, Higuchi S, Kakimoto K. Application of an intraoral scanner for dental health guidance―High precisionin vitro detection of periodontal tissue changes―. J
Osaka Dent Univ 2020; 54: 263 272.
3）Tani A, Higuchi S, Kakimoto K. Application of intraoral
scanners in dental health guidance―Clinical study onrecording accuracy and changes in the simulated display of the periodontal tissue―. J Osaka Dent Univ
2021; 55: 245 250.
4）Chiu A, Chen YW, Hayashi J, Sadr A. Accuracy of CADCAM digital impressions with different intraoral scanner parameters. Sensors 2020; 20: 1157.
5）Park J-M. Comparative analysis on reproducibility

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Prosthodont 2016; 8: 354 362.
6）Mangano FG, Veronesi G, Hauschild U, Mijiritsky E,
Mangano C. Trueness and precision of four intraoral
scanners in oral implantology―A comparative in vitro
study. PLOS ONE 2016. doi: 10.1371/journal.pone.
0163107, September 29, 2016.
7）Lee JS, Jeon YS, Strauss FJ, Jung HI, Gruber R. Digitalscanning is more accurate than using a periodontal
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8）Wang L, Chen J, Zhang W, Wu H, Yang F. Accuracy ofassessing gingival thickness in the esthetic maxillary
region by periodontal probing, cone-beam computed
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2019; 12: 10302 10309.
9）Lim HC, Lee J, Kang DY, Cho IW, Shin HS, Park JC.
Digital assessment of gingival dimensions of healthy
periodontium. J Clin Med 2021; 10: 1550.
10）Wei D, Di P, Tian J, Zhao Y, Lin Y. Evaluation of intraoral digital impressions for obtaining gingival contour inthe esthetic zone: accuracy outcomes. Clin Oral Investig
2020; 24: 1401 1410.
11）Zhang J, Huang Z, Cai Y, Luan Q. Digital assessment ofgingiva morphological changes and related factors after
initial periodontal therapy. Oral Sci 2021; 63: 59 64.
12）Ender A, Zimmermann M, Mehl A. Accuracy of complete- and partial-arch impressions of actual intraoral
scanning systems in vitro. Int J Comput Dent 2019; 22:
11 19.
13）Hotta Y. Types and mechanisms of intraoral scanners.
In: Hikita K, Baba K, eds. Introduction to the intraoral
scanner―Basics and clinical use of digital impressions.
Hotetsu Rinsho Bessatsu（Practice in Prosthodontics
Extra Issue）. Tokyo: Ishiyaku Publishers, 2019; 12 22.
（in Japanese）

ODEP Vol. 2, No. 1

72

Submission Guidelines for ODEP
1．ODEP aims to develop conservative dentistry（operative dentistry, endodontology, and periodontology）
throughthe publication of research and reviews on the following topics:（1）General dental medicine, clinical practice,and education on conservative dentistry; and（2）Conservative dentistry.
2．Papers are categorized into the following four types:（1）Original articles（reports on unique research discoveries）;（2）Reviews（discussions on research questions and objectives to indicate future directions, or summariesof the contents of existing papers to propose new ideas）;（3）Mini reviews（concise summaries of recent topics;
mini reviews include papers awarded with prizes）; and（4）Case and clinical reports（analysis of clinicalrecords useful for dental care practice and development of the field of conservative dentistry）. Reviews andmini reviews are categorized into the following:（1）Papers requested by the editorial board; and（2）Submitted
papers.
3．Original articles and case and clinical reports are limited to the following:（1）Papers that have not been publishedin other journals;（2）Papers that are not presently submitted to another journal; and（3）Papers that are notpresently scheduled for publication.
4．Acceptance or rejection of papers is determined through peer review（except for papers requested by the editorial board）.
5．Submitted papers should be concisely written in English.
6．In principle, original articles should be organized as follows:（1）Abstract;（2）Introduction;（3）Materials andMethods;（4）Results;（5）Discussion;（6）Conclusion;（7）References;（8）Figure legend;（9）Figures and Tables.
In principle, papers other than original articles should conform to the style format of original articles.
7．In principle, ODEP is published once a year in December. In addition, special issues are published when appropriate.
8．The Japanese Society of Conservative Dentistry provides a certain amount of support for the listing fees of
papers. For cases in which the lead author is not a member of the Japanese Society of Conservative Dentistry,
such support is not provided. For cases in which the lead author is a member, but the co-authors include a nonmember, partial support is provided. The cost to be borne by the authors is determined based on the numberof non-members. The costs for figures, tables and photographs, for dispatching and offprints, and for creating
the J‒STAGE registration data are borne by the authors. In the case of papers requested by the editorial
board, such costs are exempted.
9．Date of submission is the date when the submitted manuscript arrives at the secretariat of the Japanese Societyof Conservative Dentistry. Date of acceptance is the date when the reviewers determine that the submitted
manuscript can be published.
10．The listing order is the order of acceptance. A certification of publication will be issued upon request.
11．Manuscripts are to be submitted via the Japanese Society of Conservative Dentistry’s website, e-mail, or postal
mail. Manuscripts submitted for publication should be addressed to the secretariat of the Japanese Society ofConservative Dentistry.
12．In principle, authors can proofread their manuscript a maximum of two times. Extensive changes, additions, ordeletions made to the contents of the manuscript cannot be accepted. Proofs should be returned by the designated date. If the authors do not need to proofread their manuscript, they should mention this on the left sideof the cover page.
13．The copyrights for articles published in ODEP belong to the Japanese Society of Conservative Dentistry.
14．Matters not mentioned in these guidelines will be independently determined by the editorial board.
Submission of your manuscript for publication must conform to the following“Submission Guidance”as well as“Submission Guidelines.”Dec, 2022

73

The publishing charge is 10,000 yen for a Journal page including tables and figures. Extra charges for such as
figures and tables preparation, color printing of photographs will also be paid by the authors. If authors do not
pay publishing charge, the article may be retracted.

Submission Guidance（applied as of the Issue 1 of Vol. 3）
Manuscript organization
1．In principle, original articles should be organized into the following sections:（1）cover page;（2）abstract;（3）Main text（Introduction, Materials and Methods, Results, Discussion, and Conclusion）;（4）References; and（5）Figure and table captions. Page numbering should start with the cover page. In principle, manuscripts, such as
reviews or case reports, other than original articles should be organized in the same format as that of originalarticles.
2．Manuscript sections
1） Title: The title should concisely describe the contents of the manuscript. The subtitle should also clearly
describe the contents, and should not consist of only numbers.
2） Introduction: The introduction should clearly describe the background, novelty, purpose, and significance ofthe study.
3） Materials and Methods: This section should provide detailed information on the materials, equipment, or
methods used along with clear instructions so that the experiments can be reproduced by others. Parameter settings, number of specimens, extraction methods, statistics processing, and others should comply withthe purpose of the study.
4） Results: This section should simply present the findings without bias or interpretation. Measurement results
should show characteristic values including mean values and standard deviations.
5） Discussion: This section should carefully consider the materials and methods, results, and others referring
to relevant literature. Please note that it should not be overly assertive and should avoid off-topic points.
The discussion should stay focused on the study purpose and not digress into a general discussion.
6） Conclusion: The section should precisely summarize the results obtained and relate them to the purpose of
the study and hypothesis as presented in the Introduction.
3．Manuscripts should be prepared using A4-size paper. The suggested length of each typed page is 80 alphanumeric characters per line×25 lines per page using a 12-point font. Top/bottom/left/right margins should beapproximately 25 mm.

Non-Japanese names and places should be in their original names..

4．For the manuscript style, refer to the latest issue of the journal.
Ethics Code
1．Each report on the result of clinical research（clinical trial or observational research）or research involving any
specimen collected from a human body must include a clear statement that it was approved by the head of theaffiliated institution or by the research ethics review board assigned by the institution’s head, in order toexpressly indicate that the research was conducted in compliance with all applicable guidelines and laws,
including the Declaration of Helsinki and the medical research guidelines, etc. issued by the Ministry of Health,Labour and Welfare including the following:
1）Ethical Guidelines for Life Science and Medical Research Involving Human Subjects;
2）Guidelines for Gene Therapy and Other Clinical Research; and3）Clinical Trials Act.
2．Each report on the result of research on, or a case concerning, regenerative medicine technology, etc. as definedODEP Vol. 2, No. 1

74

in the Act on the Safety of Regenerative Medicine must include a clear statement that the technology was provided to the patients in compliance with the aforementioned Act.
3．Each report on a case of a therapeutic method involving any off-label drug or device, or any pharmaceutical,
medical device, regenerative medicine, or other related product not domestically approved must include a clear
statement that the use was approved by the committee concerned（research ethics review board, review
board for unapproved new drugs, etc.）
at the affiliated institution or that the case report was approved for publication by ODEP’s Clinical and Epidemiological Ethics Committee.
4．In each instance of publication of an academic paper, all personal information must be thoroughly protected sothat none of the research subjects（patients）can be identified from it.
5．In each instance where a patient’s clinical photo or X-ray image is included in an academic paper for publication,a clear statement must be provided that the consent of each such patient（or a parent, guardian, or proxy if thepatient is a minor or in case it is otherwise difficult to obtain consent from the patient）was duly obtained.
6．Each report on the result of research involving animal subjects must include a clear statement that the research
was approved by the animal experiment committee, etc. at the affiliated institution.
Cover page
1．The title, authors’ names, institutional affiliations, and corresponding author’s contact address should be centered,with a new line for each item, on the cover page.
2．The title should be in upper and lowercase letters, where the first letter of each word is uppercased and theremaining letters are lowercased. Articles, prepositions, conjunctions, and commonly used technical terms arelowercased. For hyphenated compound words, the letters following the hyphen should be lowercased.
3．The corresponding author’s contact details should include the following information: one author’s name, institutional affiliation, postal address, telephone and fax numbers, and e-mail address.
Abstract
1．The abstract should be a maximum of 400 words organized into four sections with the following headings: Purpose, Methods, Results, and Conclusion. Approximately three keywords should be placed at the end of theabstract.
2．Contributors should put considerable effort into preparing the abstract as it may determine whether or not thereader continues with the manuscript. When necessary, abstracts should be checked by a native English
reviewer（preferably with expertise in dental medicine）.
Main text
1．Introduction, Materials and Methods, Results, Discussion, and Conclusion are the main headings and are notnumbered.
2．Subparagraphs should be numbered in the following order: 1. 2. 3. …; 1）2）3）…;（1）（2）（3）…; ① ② ③ …;
and a. b. c. …
3．English characters should be written in the following manner:
1） Generally, only the last name should be used to indicate a person.
2） When the name of a product or manufacturer must be in the original language, the first letter of each word
should be uppercased and the remaining letters lowercased.
In principle,“generic name（product name, company name, city［state in the case of U.S.］, country）”
shouldbe used in English manuscripts. Trademark and registration symbols ® and TM are not required.
3） Regarding common nouns in German or Latin, the first letter should be uppercased and the remaining letters lowercased. For common nouns in English and French, all letters should be lowercased.
4） Regarding binominal nomenclature, the first letter of the genus should be uppercased and the remaining
letters lowercased. The names of all genera and species should be italicized. When the same genusDec, 2022

75

appears frequently, it is acceptable to replace the name with the initial after the first use.
Example: Streptococcus mutans → S. mutans
5） For nouns that must be in their original language, other than German, Latin, English or French, all lettersshould be lowercased, except for commonly used technical terms.
4．In principle, SI units are used for measurements.
5．Any conflicts of interests（COI）must be declared after the conclusions. When there is no COI, the statement“The authors declare no conflict of interest related to this paper”should be included.
6．Acknowledgments for all sources supporting this study including grant funds should be added after of COI.
References
1．References must be listed at the end of the main text, and numbered in the same order as they appear in thetext.
2．In the main text, a cited reference should appear with a superscript numeral and closing parenthesis. When two
references are cited, a comma should be used to separate them; more than two references should be connectedby an en dash between the first and last numeral.
Examples:“by authors3）”,“…is reported7,8）”,“previous studies10‒15）show”
3．Examples of Reference
a ．Journal articles
Number）Last name and first name of all authors with a comma separating each author. Title of paper. Name ofjournal and publishing year in the Christian era; Volume number: Inclusive page numbers of paper.
Example:1）Clark AB, Erickson D, Hamilton FG. Tensile bond strength and modulus of elasticity of several composite resins. J Dent
Res 1992; 37: 618‒621.

b ．Book
Number）Author（co-authors）. Title of book. First/last volume. Edition. Publisher’s name: Publisher’s location（City）; Publishing year in the Christian era. Cited pages.
Example:2）Phillips RW. Skinner’s science of dental materials. 9th ed. WB Saunders: Philadelphia; 1991. 219‒221.

c ．Book with co-authors
Number）Contributor’s name. Title of contributed article. Name of editor（editor-in-chief）. Book title. First/lastvolume. Edition. Publisher’s name: Publisher’s location（City）; Publishing year in the Christian era.
Cited pages.
Example:3）Torneck CD. Dentin-pulp complex. Ten Cate AR. Oral histology. 5th ed. Mosby: St. Louis; 1998. 150‒196.

For cases in which each author’s contribution is not separately indicated, the author’s name and the title of thecontributed article should not be listed.
Number）Name of editor（editor-in-chief）. Title of book. First/last volume. Edition. Publisher’s name: Publisher’slocation（City）; Publishing year in the Christian era. Cited pages.
d ．Other writing styles
・Abstracts of scientific meeting
Number）Presenter（all presenters should be cited. A comma should be used to separate the authors in the caseof co-presentations.）. Title of abstract. Name of journal and publishing year in the Christian era; Volume number: Inclusive page numbers, abstract number.
Example:4）Marais JT. Cleaning efficacy of a new root canal irrigation material. J Dent Res 1998; 77: 669, Abst. No. 300.

・ Journal articles in press
In principle, the same style as that of a regular journal article should be used; however, when the inclusive page

ODEP Vol. 2, No. 1

76

numbers are not known, they can be omitted. The statement“in press”should be shown at the end.
Example:5）Sato K. Effect of toothbrushes on gingival abrasion. J Periodont Res 1994; 29: in press.

・Electronic journal
In principle, the same style as that of a regular journal article should be used; however, when the inclusive page
numbers are not known, the DOI and other indicators should be shown. When papers are published in electronic journals before printing, the statement［Epub ahead of print］should be shown after the publishing year
and month.
Example:6）Sunada N, Ishii R, Shiratsuchi K, Shimizu Y, Tsubota K, Kurokawa H, Miyazaki M. Ultrasonic measurement of theeffects of adhesive application and power density on the polymerization behavior of core build-up resins. Acta Odontol Scand; doi: 10.3109/00016357.2011.654252

・Internet website
Page publisher. Title of page. URL address.（Access date）
Example:7）World Health Organization. Continuous improvement of oral health in the 21st century. http://www.who.int/oral_
health/en/（cited 2005. 10. 1）

4．In principle, journal names should be abbreviated in accordance with the format used by the journal.
Figures and Tables
1．Figures, photographs, and tables are categorized into figures and tables, and then numbered. Paper size shouldbe A4, and each figure and table should be printed on a separate page. The numbers allocated for the figures
and tables should be consistent with those referred to in the text.
2．Figures and tables should be accompanied by explanations that are easily understandable. Explanations of figures/tables are presented as captions and explanations for tables/tables should be presented as footnotes.
3．If authors wish to have the figures shown in color, color data should be attached; if authors wish to have thefigures shown in black and white, black-and-white data should be attached.
Notes on creating imaging data:
・ The jpg data format should be used if possible.
・ Image size should correspond to the layout; image resolution should be at least 300 dpi for photographs andat least 1,200 dpi for line drawings.
Sending manuscript intended for publication
1．The manuscript（cover page, abstract, main text, references, and figure and table captions are created as onefile）should be formatted as a Microsoft Office Word（hereafter“Word”）document.
2．Figures should be provided in jpg or pdf format.
3．Tables should be provided in Microsoft Office Excel, jpg, pdf, or Word format.
4．File titles should be as follows:“author name”_“university name（name after department is not needed）”_“manuscript/figure/table/submission form”_“.filename extension（indicating file type）”.
Example）Nihon Tarou_Nihon University/manuscript.docx; Nihon Taro_Nihon University_Figure.jpg; Nihon
Taro_Nihon Univesity_Table.xlsx; Nihon Taro_Nihon University_submission form.pdf
Also, pdf files with embedded fonts for all contents are acceptable for submission. In such cases, file names
should be as follows: Nihon Taro_Nihon University_comprehensive manuscript.pdf
5．E-mail title（subject）should be“Submitted papers for ODEP”.
6．Submitted papers should be sent to the e-mail address of Oral Health Association of Japan: hensyu6@kokuhoken.
or.jp. For safety, also send as a CC to hensyu5@kokuhoken.or.jp.
7．When e-mail submission is difficult for such reasons as the file size is too large, submission via an FTP serverDec, 2022

77

and other methods are acceptable. In such cases, the intent of submission should be informed by e-mail. In thee-mail, information regarding the site and other methods for downloading the file should be provided.
8．The submission form of ODEP provided on the Japanese Society of Conservative Dentistry’s home page（http://www.kokuhoken.or.jp/exterior/ jscd/fileform/）can be used for submission.

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The Japanese Society of Conservative Dentistry authorized Japan Academic Association For Copyright
Clearance（JAC）to license our reproduction rights of copyrighted works. If you wish to obtain permissionsof these rights, please refer to the homepage of JAC（http://www.jaacc.org/en/）and confirm appropriateorganizations to request permission.

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